Co-packaging to mitigate intermolecular recombination

ABSTRACT

The subject matter disclosed herein is generally directed to methods and compositions for stable transduction of target cells with libraries of genetic elements. The invention reduces intermolecular recombination between library elements and integration of multiple genetic elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/627,183, filed Feb. 6, 2018. The entire contents of theabove-identified application are hereby fully incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.HG009283 and HG006193 granted by National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD_2465WP_ST25.txt”,301 KB, created on Jan. 30, 2019) is herein incorporated by reference inits entirety.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed to methods andcompositions for stable transduction of target cells with libraries ofgenetic elements. The invention reduces intermolecular recombinationbetween library elements and integration of multiple genetic elements.

BACKGROUND

Lentiviral vectors provide a convenient, scalable platform to delivergenetic perturbations to cells en masse and read out the identity ofeach perturbation by next-generation sequencing^(1,2). Certain screenmodalities rely on the delivery of multiple sequences per lentiviralvector in order to probe gene interactions with combinations ofperturbations or to encode the identity of each perturbation in aneasily-detectable barcode sequence, such as in CRISPR-based single-cellgene expression screens³⁻⁸. However, these methods are highlysusceptible to intermolecular recombination that scrambles engineeredassociations between the variable sequences. For screens where allvariable elements are sequenced directly (e.g. targeted pairs of geneknockouts), recombination events can be detected and filtered out beforestatistical analysis^(6, 9). However, in situations where certainfunctional sequences are not read out, such as when a barcode stands inas a proxy for a genetic perturbation, recombination can lead tomislabeled data and has been noted to decrease the statistical power ofgenetic screens at a given number of cells analyzed^(10, 11).

Intermolecular recombination can arise from the template-switchingactivity of the lentiviral reverse-transcriptase¹⁶. As the lentiviruscapsid normally packages a dimer of RNA genomes, the effect persistseven under dilute conditions where cells are infected by a singlevirion. The fraction of cells with recombined integrants depends on thedistance between variable sequences and has been measured to exceed 30%for distances of >1 kb, which may occur when the sequences are separatedby regulatory elements such as promoters, or used as 3′ barcodes in anexpressed transcript, where recombination events can lead to aneffective scrambling of barcodes and perturbations, which may bereferred to herein as barcode swapping¹⁰⁻¹².

SUMMARY

The invention provides improved lentiviral or retroviral systems withreduced intermolecular recombination between library elements andreduced integration of multiple genetic elements in a target cell.

In one aspect, the invention provides a non-naturally occurringlentiviral or retroviral system comprising a polynucleotide having atleast a first engineered association and a second engineeredassociation, wherein the system has reduced recombination activity, ortemplate switching activity, or multiple integration activity.

In an embodiment, the engineered system comprises an inhibitor ofrecombination activity, or template switching activity, or multipleintegration activity. In an embodiment, the inhibitor of templateswitching is a carrier polynucleotide. The carrier polynucleotide can beinvolved in or affect any aspect of lentiviral packaging, and functionsto reduce recombination activity or template switching activity, ormultiple integration. For example, in an embodiment of the invention,the carrier polynucleotide is packaged with or forms a heterodimer withthe polynucleotide comprising the one or more engineered associations,but lacks sufficient homology such that recombination activity, templateswitching activity, or multiple integration activity is reduced oreliminated. In an embodiment of the invention, the reduction inrecombination activity, template switching activity, or multipleintegration activity can be 2×, 5×, 10×, 20×, 50×, 100×, 500×, 1000× orgreater as compared to packaging without the carrier polynucleotide. Inpackaging reactions, carrier polynucleotides are usually in excess. Incertain embodiments, the carrier polynucleotide to payloadpolynucleotide ratio in packaging is from 5:1 to 10:1 or from 10:1 to20:1 or from 20:1 to 50:1, or from 50:1 to 100:1 or from 100:1 to 500:1,of from 500:1 to 1000:1 or greater.

In another embodiment, the inhibitor of recombination activity, ortemplate switching activity, or multiple integration activity can be anycarrier polynucleotide transfected into a packaging cell and presentwith the payload to be packaged, which carrier polynucleotide is notdesigned to be packaged. Such carriers include, without limitation,single and double stranded DNA, replicable and non-replicable plasmidtype vectors, including prokaryotic and eukaryotic vectors. In anon-limiting example set forth herein, bacterial plasmid pUC19, whichdoes not replicate in a packaging cell, is not transcribed, and is notdesigned to be packaged in a lentiviral particle, is demonstrate toinhibit recombination activity, template switching activity, or multipleintegration activity.

In an embodiment, the inhibitor of recombination activity, or templateswitching activity, or multiple integration activity comprises apolynucleotide designed to hybridize with all or part of the 5′ UTR,including but not limited to such regions as US-PBS complex or the dimerinitiation site (DIS).

In an embodiment, recombination activity, template switching activity,or multiple integration activity is reduced by rearranging elements ofthe payload polynucleotide. This includes without limitation, deletionof 5′ UTR elements and/or introduction of 5′ UTR elements elsewhere inthe sequence of the payload to be packaged. In an embodiment,introduction and/or relocation of the DIS provides lentivirus genomes(e,g., payloads) that package predominantly or completely as monomers.

In certain embodiments of the invention, recombination activity,template switching activity, or multiple integration activity ismodulated by altering interaction of the payload with the capsid. In oneembodiment, the lentivirus nucleocapsid (NC) protein is altered bymutating the zinc-finger region so as to disrupt NC-dependentdimerization.

In an embodiment of the invention, the system comprises a multiplicityof payload polynucleotides, each having at least a first engineeredassociation and a second engineered association. The multiplicity ofpolynucleotides can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more and furtherany number of polynucleotides each having at least a first engineeredassociation and a second engineered association.

In an embodiment of the invention, the first engineered associationcomprises a genetic perturbation. In an embodiment of the invention,both the first and the second engineered association each comprises agenetic perturbation. In an embodiment of the invention, the firstengineered association comprises a genetic perturbation and the secondengineered association comprises an identifier, such as but not limitedto a unique molecular identifier. In an embodiment, the unique molecularidentifier is a barcode.

In an embodiment of the invention, the carrier polynucleotide comprisesor encodes non-recombinogenic RNA sequences or proteins that are capableof dimerizing with the polynucleotide having engineered associations. Incertain embodiments, the RNA sequences or proteins disrupt recombinationwith the polynucleotide having engineered associations.

According to the invention the reduced recombination or templateactivity comprises reduced hairpin formation or dimerization throughmodification, knockdown or knockout of retroviral genomic RNA orretroviral protein involved in dimerization.

Further, in certain embodiments, the modification, knockdown or knockoutof the retroviral genomic RNA retroviral protein comprises modification,knockdown or knockout of nucleocapsid (NC)-protein(s) or RNA forexpression thereof or modification, knockdown or knockout of stem-loop Ielement (SLI) element or modification, knockdown or knockout of genomicRNA whereby U5:AUG pairing is prevented or modification, knockdown orknockout of a dimer initiation site (DIS).

In an embodiment of the invention, the polynucleotide sequence encodingone or more genetic perturbations encodes an over expressed gene, anRNAi based system, a zinc finger nuclease, a transcriptionactivator-like effector nuclease (TALEN), a meganuclease, or aCRISPR-Cas system.

In another embodiment, the sequence encoding one or more geneticperturbations encodes a CRISPR-Cas9 system. In another embodiment, thesequence encoding one or more genetic perturbations encodes one or moreguides.

In an aspect, the invention provides a method of preparing a lentiviralor retroviral system comprising a polynucleotide having at least a firstengineered association and a second engineered association wherein thesystem has reduced recombination activity or template switchingactivity, or multiple integration activity. In an embodiment of theinvention, the reduction can be 2×, 5×, 10×, 20×, 50×, 100×, 500×, 1000×or greater. In an embodiment, the method comprises packaging thepolynucleotide with an inhibitor of template switching.

In an embodiment, the method comprises packaging the polynucleotide witha carrier polynucleotide. As set forth above, the carrier polynucleotidecan be involved in or affect any aspect of lentiviral packaging andfunctions to reduce recombination activity or template switchingactivity, or multiple integration. In an embodiment of the invention,the method comprises packaging of a multiplicity of polynucleotides,each having at least a first engineered association and a secondengineered association. The multiplicity of polynucleotides can be 2, 3,4, 5, 6, 7, 8, 9, 10 or more and further any number of polynucleotideseach having at least a first engineered association and a secondengineered association.

In an embodiment of the invention, the method comprises geneticperturbation. In an embodiment of the invention, both the first and thesecond engineered association each comprises a genetic perturbation. Inan embodiment of the invention the first engineered associationcomprises a genetic perturbation and the second engineered associationcomprises an identifier, such as but not limited to a unique molecularidentifier. In an embodiment, the unique molecular identifier is abarcode. In an embodiment of the invention the method comprises use ofnon-recombinogenic RNA sequences or proteins that are capable ofdimerizing with the polynucleotide having engineered associations.

According to the invention, the reduced recombination or templateactivity comprises reduced hairpin formation or dimerization throughmodification, knockdown or knockout of retroviral genomic RNA orretroviral protein involved in dimerization. Further, in certainembodiments, the modification, knockdown or knockout of the retroviralprotein comprises modification, knockdown or knockout of nucleocapsid(NC)-protein(s) or RNA for expression thereof or modification, knockdownor knockout of stem-loop I element (SLI) element or modification,knockdown or knockout of genomic RNA whereby U5:AUG pairing is preventedor modification, knockdown or knockout of a dimer initiation site (DIS).

In an aspect, the invention provides a method of preparing a lentiviralor retroviral system comprising a polynucleotide having at least a firstengineered association and a second engineered association wherein thesystem has reduced recombination activity or template switchingactivity, or multiple integration activity. In an embodiment of theinvention, the reduction can be 2×, 5×, 10×, 20×, 50×, 100×, 500×, 1000×or greater. In an embodiment, the method comprises packaging thepolynucleotide with an inhibitor of template switching.

In an embodiment, the method comprises packaging the polynucleotide witha carrier polynucleotide. As set forth above, the carrier polynucleotidecan be involved in or affect any aspect of lentiviral packaging andfunction to reduce recombination activity or template switchingactivity, or multiple integration activity. In an embodiment of theinvention, the method comprises packaging of a multiplicity ofpolynucleotides, each having at least a first engineered association anda second engineered association. The multiplicity of polynucleotides canbe 2, 3, 4, 5, 6, 7, 8, 9, 10 or more and further any number ofpolynucleotides each having at least a first engineered association anda second engineered association.

In an embodiment of the invention, the method comprises a geneticperturbation. In an embodiment of the invention, both the first and thesecond engineered association each comprises a genetic perturbation. Inan embodiment of the invention, the first engineered associationcomprises a genetic perturbation and the second engineered associationcomprises an identifier, such as but not limited to a unique molecularidentifier. In an embodiment, the unique molecular identifier is abarcode.

In an embodiment of the invention the method comprises use ofnon-recombinogenic RNA sequences or proteins that are capable ofdimerizing with the polynucleotide having engineered associations.According to the invention, the reduced recombination or templateactivity comprises reduced hairpin formation or dimerization throughmodification, knockdown or knockout of retroviral genomic RNA orretroviral protein involved in dimerization.

Further, in certain embodiments, the modification, knockdown or knockoutof the retroviral protein comprises modification, knockdown or knockoutof nucleocapsid (NC)-protein(s) or RNA for expression thereof ormodification, knockdown or knockout of stem-loop I element (SLI) elementor modification, knockdown or knockout of genomic RNA whereby U5:AUGpairing is prevented or modification, knockdown or knockout of a dimerinitiation site (DIS).

Screening using the CRISPR technology and method and systems of theinvention is particularly advantageous because of its simplicity,specificity and versatility. For example genome-wide GeCKO and SAMlibraries target every gene in the mouse or human genes and knock-out ortranscriptionally activate each gene. Alternatively, libraries may bepathway-focused and screened under specific conditions such as bypositive or negative selection, to identify important genes in apathway. In an embodiment, a population of cells may be transfected witha library to knock out or activate certain genes, transfectants ofinterest identified on the basis of phenotypic screens, and cellproducts of the transfection identified by a unique molecular identifieroriginally associated with each gene knocked out, knocked down oractivated. In certain embodiments, phenotypic screens identify geneexpression profiles which may then be associated with an originaltransfectant. Generally in such embodiments, genetic elements for knockout, knock down, or activation are each associated in the library withan identifier, which can be but is not limited to a unique molecularidentifier such as a barcode.

Lentiviral packaged libraries include particles containing heterodimersand recombinant heterodimers. Packaged heterodimers occur, for example,when two or more library members are contained in one cell of apackaging cell line and is accompanied by recombination or templateswitching in of the heterodimer. For example, a targeting library may beconstructed such that in each library member, a gene targeting sequencesuch as a guide sequence of a CRISPR system is separated to some degreefrom an identifier element such as a barcode, but the interveningsequence is the same, and promotes recombination between library memberswhen dimerized. In certain embodiments, the sequence interveningsequence common to the library members corresponds to the direct repeatthat binds to a CRISPR protein. Recombination produces mispairing ofguide sequences with barcodes, hence degrades information obtainablefrom the screen. The lentiviral systems described herein minimizerecombination, providing lentivirus packages that are effectivelymonomeric. By “effectively monomeric” is meant that a library member ispackaged as a monomer or in a manner that reduces or eliminatesrecombination. In certain embodiments, a library member, which is apolynucleotide having at least a first engineered association and asecond engineered association, is packaged with a nucleic acid that isnot recombinogenic, referred to herein as a stuffer. In certainnon-limiting embodiments, a stuffer nucleic acid lacks any substantialhomology with the polynucleotide having the first and second engineeredassociation. In certain embodiments, a nucleic acid is provided in apackaging cell that is not packaged but reduces heterodimers andrecombination thereof. The nucleic acid can be any replicable vectorthat need not produce a packageable polynucleotide. In an embodiment ofthe invention, the vector is pUC19.

Certain evidence has suggested that lentiviral genome dimerizationnormally occurs after RNA is packaged and virus particles are released.For example, 70S RNA dimers could not be isolated from infected cellsand viral particles harvested upon formation contained monomeric RNAwhich dimerized minutes or hours after particle release. Also,dimerization of the RNA in the particles was blocked if the virus wassolubilized with detergent. (Canaani et al., 1973, Proc. Natl. Acad.Sci. USA 72:401-405). In certain models, NC protein contributes todimerization. For example, in one model, the NC protein after releasefrom the Gag polyprotein, binds to each RNA and unfolds double-strandedstructures near the 5′ ends, allowing interstrand contacts to form. Inthis regard, there are observations that prevention of gag polyproteincleavage by protease inactivation or mutation of NC results in virusparticles that contain monomeric RNA. (Oertle and Spahr, 1990; Stewartet al., 1990, J Virol 64:5076-92; Dupraz et al., 1990).

Certain key nucleotides involved in the RNA dimerization event make up apalindromic sequence between the PBS and the major splice donor, and RNAsequences on both sides of this palindrome can form a stem-loopstructure with the palindrome in the hairpin loop. Deletion of thisstem-loop motif completely abolished dimerization of the 1 HIV-1 RNAfragment in vitro. Skripkin et al., 1994, Proc Natl Acad Sci USA91:4945-4949.

Further, duplication of the DIS/DLS region in viral RNA causesproduction of virus particles containing partially monomeric RNAswithout modifying any viral proteins and yields particles comparable incertain aspects to wild-type particles. Sakuragi et al., 2002, J. Virol.76:959-967. The results indicate that RNA dimerization is not requiredfor viral RNA packaging, virion maturation, and reverse transcription.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIG. 1 —Schematic of delivery of barcoded lentiviral plasmid libraryinto target cells. Viral genomes containing sgRNAs and transcribed RNAbarcodes, driven by U6 and EF1a promoters, are packaged into virions andintegrated into target cells. Dimeric packaging of library plasmids mayyield homodimeric or heterodimeric library associations or, in the caseof cow packaging with a non-homologous carrier lentivirus (purple), afunctionally monomeric virion. Virions with two different libraryelements have the capacity for recombination between sgRNAs and barcodesas well as potential for integration of multiple perturbations into thetarget cell, whereas co-packaging with a non-homologous vector limitsthese alternatives.

FIG. 2 —An example method of constructing viral libraries using themethods described herein.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2^(nd) ed., J. Wiley & Sons (New York, N.Y. 1994),March, Advanced Organic Chemistry Reactions, Mechanisms and Structure4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofkerand Jan van Deursen, Transgenic Mouse Methods and Protocols, 2^(nd)edition (2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +1-10% or less, +/−5% or less,+/−I % or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

As used herein, “engineered association” means a library member portionthat comprises an engineered structural and/or functional part,including but not limited to a guide sequence for a CRISPR system, or atag or identifier such as a unique molecular identifier (UMI) or barcodeor other tracking element. The engineered structural or functional partis physically associated with the library member in that it is linked tonucleotides or other chemical parts of the library member. Two or moreengineered associations are linked when they are comprised by a singlepolynucleotide or other monomeric molecule. A library polynucleotidecomprising engineered associations may be referred to as a “payload” or“template.”

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” or “an example embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the invention. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Overview

Retroviral systems, including lentivirus-based systems, can bepseudo-diploid, that is two viral genomes are packaged into each viralparticle and are non-covalently linked. During viral genome replication,the reverse transcriptase can switch from one template to another whenit synthesizes a DNA provirus from a dimeric RNA genome, and thisprocess happens most frequently at homologous regions. The frequency ofrecombination may depend on the distance between two regions which hasbeen estimated to be 2% every kilobase. Thus, when libraries of distinctvector sequence are packaged together, template switching could lead torecombination that randomly shuffles associations between sequences,such as associations between sequences encoding one or more geneticperturbations and unique molecular sequence, for example a uniquemolecular sequence that identifies the encoding perturbation.

Embodiments disclosed herein provide retroviral systems comprisingmodifications that mitigates those effects by reducing recombination ortemplate switching activity. Further included are modified methods forretroviral vector packaging. The modified retroviral systems disclosedherein may be used for combinatorial screening of perturbations,including single cell screening.

Engineered Viral Systems

The present disclosure includes non-naturally engineered viral systems.In some examples, the non-naturally occurring engineered viral systemsmay be a lentiviral or retroviral system. The systems disclosed hereinmay comprise a first polynucleotide having at least a first and secondengineered association. For ease of reference, the remaining disclosurewill address systems with a first and second engineered association, butmore than two engineered associations are also envisioned. One or moreactivities of the engineered systems may be reduced (e.g., as comparedto a non-engineered counterpart system). Such activities may includerecombination activity, or template switching activity, and multipleintegration activity.

The engineered systems herein may comprise a multiplicity ofpolynucleotides. In certain embodiments, the retroviral system maycomprise a multiplicity of first polynucleotides. The multiplicity offirst polynucleotides may comprise different combinations of engineeredassociations. As used herein, the term “retroviral” is intended toencompass both retroviral and lentivirus-based systems. The first andsecond engineered association represent sequences that need to remainassociated with one another throughout the life cycle of thepolynucleotide. For example, the polynucleotide may be a vector and thefirst and second association encode elements that need to remainassociated on the same polynucleotide for further downstreamapplications. In certain example embodiments, the first and engineeredassociations may be located 1 kb or greater apart on the polynucleotidesequence. In certain example embodiments, the engineered associationsmay be located 2 kb or greater apart on the polynucleotide sequence.

The retroviral system may comprise an inhibitor of recombination ortemplate switching. In certain example embodiment, the retroviral systemmay further comprise a second polypeptide. The second polynucleotide maybe a carrier polynucleotide comprising non-recombinogenic RNA sequencesor sequences with limited homology to the first nucleotide or otherwiseconfigured to impair or prevent homologous recombination with the firstpolynucleotide when packaged together within a viral particle. Inanother embodiment, the second polynucleotide may result in reducedhairpin formation or dimerization through modification, knockdown orknockout of retroviral genomic RNA or retroviral proteins involved indimerization.

In certain example embodiments, the second polypeptide may be 2 kb to 10kb in size. In certain example embodiments, the second polypeptide is2.0 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb,2.9 kb, 3.0 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb,3.8 kb, 3.9 kb, 4.0 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb,4.7 kb, 4.8 kb, 4.9 kb, 5.0 kb, 5.1 kb, 5.2 kb, 5.3 kb, 5.4 kb, 5.5 kb5.6 kb, 5.7 kb, 5.8 kb, 5.9 kb, 6.0 kb, 6.1 kb, 6.2 kb, 6.3 kb, 6.4 kb,6.5 kb, 6.6 kb, 6.7 kb, 6.8 kb, 6.9 kb, 7.0 kb, 7.1 kb, 7.2 kb, 7.3 kb,7.4 kb, 7.5 kb, 7.6 kb, 7.7 kb, 7.8 kb, 7.9 kb, 8.0 kb, 8.1 kb, 8.2 kb,8.3 kb, 8.4 kb, 8.5 kb, 8.6 kb, 8.7 kb, 8.8 kb, 8.9 kb, 9.0 kb, 9.1 kb,9.2 kb, 9.3 kb, 9.4 kb, 9.5 kb, 9.6 kb, 9.7 kb, 9.8 kb, 9.9 kb, or 10.0kb in size.

In certain example embodiments, the second polypeptide may be selectedto have less than 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%,49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%,35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%,21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1% complementarity to the first polynucleotide.

In certain example embodiments, the second polypeptide is a lentiviralvector. In certain example embodiments, the lentiviral vector has longterminal repeat to long terminal repeat distance (LTR-LTR distance) of2.5 kb, 2.4 kb, 2.3 kb, 2.2 kb, 2.1 kb, 2.0 kb, 1.9 kb, 1.8 kb, 1.7 kb.1.6 kb, 1.5 kb, 1.4 kb, 1.3 kb, 1.2 kb. 1.1 kb, or 1.0 kb.

In certain example embodiments, the lentiviral vector comprises one ormore LTR mutations in one or both LTR regions that abrogate integrationcapability.

Other factors that may be considered in selecting or designing secondpolynucleotide include GC content, presence/absence of repeats, sequencesignatures that affect DNA helix parameters, using supercoiled versusrelaxed plasmids, nicked or un-nicked plasmids, methylated ornon-methylated plasmids.

Retroviral Systems

The viral backbone of the retroviral system may be any retrovirussuitable for use in delivering expression constructs to cells. Exampleretroviral systems include moloney murine leukemia virus (MoMuLV),feline immunodeficiency virus (FIV), HIV-1 based packaging systems(HIV), and lentiviral based systems. In certain example embodiments, theretroviral system is based on Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), gibbon ape leukemia virus (GaLV) human immunodeficiency virus(HIV) and Rous Sarcoma Virus (RSV). (see, e.g., Buchscher et al, J.Virol. 66:2731-2739 (1992); Johann et al, J. Virol. 66: 1635-1640(1992); Sommnerfelt et al, Virol. 176:58-59 (1990); Wilson et al, J.Virol. 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

Vectors that are based on HIV may retain <5% of the parental genome, and<25% of the genome may be incorporated into packaging constructs, whichminimizes the possibility of the generation of revertantreplication-competent HIV. The vector region may include sequences formthe 5′ and 3′ LTRs of a lentivirus. In some instances, the vector domainincludes the R and U5 sequences from the 5′ LTR of a lentivirus and aninactivated or self-inactivating 3′ LTR from a lentivirus. The LTRsequences may be LTR sequences from any lentivirus from any species. Forexample, they may be LTR sequences from HIV, SIV, FIV or BIV. Wheredesired, the packaged viral barcoded library may be made up of selfinactivating vectors that contain deletions of the regulatory elementsin the downstream long-terminal-repeat sequence, eliminatingtranscription of the packaging signal that is required for vectormobilization. As such, the vector region may include an inactivated orself-inactivating 3′ LTR. The 3′ LTR, may be made self-inactivating byany convenient method. For example, the U3 element of the 3′ LTR maycontain a deletion of its enhancer sequence, such as the TATA box. Sp1and NF-kappa B sites. As a result of the self-inactivating 3′ LTR, theprovirus that is integrated into the host cell genome will comprise aninactivated 5′ LTR. Optionally, the U3 sequence from the lentiviral 5′LTR may be replaced with a promoter sequence in the viral construct.This may increase the titer of virus recovered from the packaging cellline. An enhancer sequence may also be included. In certain aspects, theviral construct is a non-integrating lentiviral construct, where theconstruct does not integrate by virtue of having a defective (e.g., bysite-specific mutation) or absent integrase gene. Integrate-detectivelentiviral vectors are described, e.g., in Banasik and McCray (2010)Gene Therapy 17(2):150-157.

In certain example embodiments, a lentivirus based system is used.Lentiviruses are members of the retrovirus family. Widely usedretroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereof(see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); Johann etal, J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al, Virol. 176:58-59(1990); Wilson et al, J. Virol. 63:2374-2378 (1989); Miller et al, J.Virol. 65:2220-2224 (1991); PCT/US94/05700).

The embodiments disclosed herein may also be useful in non-retroviralbased systems, that are pseudo-diploid or otherwise known to have thesame recombination and template-switching limitations of lentivirus andretrovirus systems disclosed herein.

Carrier Polynucleotides

The invention provides inhibitors of recombination activity, templateswitching activity, or multiple integration activity. In some cases, theengineered systems described herein comprise an inhibitor of templateswitching. In an embodiment, the inhibitor of template switching is acarrier polynucleotide. The carrier polynucleotide can be involved in oraffect any aspect of lentiviral packaging, and functions to reducerecombination activity or template switching activity, or multipleintegration. For example, in an embodiment of the invention, the carrierpolynucleotide is packaged with or forms a heterodimer with thepolynucleotide comprising the one or more engineered associations, butlacks sufficient homology such that recombination activity, templateswitching activity, or multiple integration activity is reduced oreliminated. In an embodiment of the invention, the reduction inrecombination activity, template switching activity, or multipleintegration activity can be 2×, 5×, 10×, 20×, 50×, 100×, 500×, 1000× orgreater as compared to packaging without the carrier polynucleotide. Inpackaging reactions, carrier polynucleotides are usually in excess. Incertain embodiments, the carrier polynucleotide to payloadpolynucleotide ratio in packaging is from 5:1 to 10:1 or from 10:1 to20:1 or from 20:1 to 50:1, or from 50:1 to 100:1 or from 100:1 to 500:1,of from 500:1 to 1000:1 or greater.

In another embodiment, the inhibitor of recombination activity, ortemplate switching activity, or multiple integration activity can be anycarrier polynucleotide transfected into a packaging cell and presentwith the payload to be packaged, which carrier polynucleotide is notdesigned to be packaged. Such carriers include, without limitation,single and double stranded DNA, replicable and non-replicable plasmidtype vectors, including prokaryotic and eukaryotic vectors. In anon-limiting example set forth herein, bacterial plasmid pUC19, whichdoes not replicate in a packaging cell, is not transcribed, and is notdesigned to be packaged in a lentiviral particle, is demonstrate toinhibit recombination activity, template switching activity, or multipleintegration activity.

In some embodiments, the carrier polynucleotide comprises or encodes oneor more non-recombinogenic RNA sequences. Alternatively or additionally,the carrier polynucleotide may encode proteins that capable ofdimerizing with the polynucleotide having engineered association.

Reduced recombination or template activity herein may comprise reducedhairpin formation or dimerization through modification, knockdown orknockout of retroviral genomic RNA or retroviral protein involved indimerization. In some examples, the retroviral genomic RNA or retroviralprotein comprises nucleocapsid (NC)-protein(s) or RNA encoding thereof,stem-loop I element (SLI), genomic RNA in which U5:AUG pairing isprevented, a dimer initiation site (DIS), or any combination thereof.

In an embodiment, the inhibitor of recombination activity, or templateswitching activity, or multiple integration activity comprises apolynucleotide designed to hybridize with all or part of the 5′ UTR,including but not limited to such regions as U5-PBS complex or the dimerinitiation site (DIS). In an embodiment, the inhibitor polynucleotidecan be RNA produced concurrently with the payload, or added to thepayload prior to packaging. In an embodiment, the inhibitorpolynucleotide can be synthetic. Tran et al., 2015, Retrovirology 12:83reviews conserved determinants of lentiviral genome dimerization.

In an embodiment, recombination activity, template switching activity,or multiple integration activity is reduced by rearranging elements ofthe payload polynucleotide. This includes without limitation, deletionof 5′ UTR elements and/or introduction of 5′ UTR elements elsewhere inthe sequence of the payload to be packaged. In an embodiment,introduction and/or relocation of the DIS provides lentivirus genomes(e,g., payloads) that package predominantly or completely as monomers.Sakuragi et al., 2002, J. Virol. 76:959-967 reports several HIV mutantscomprising multiple and rearranged copies of viral E/DLS sequences.According to the invention, 5′ UTR elements can be added and/orrearranged in payload genomes, taking care not to interrupt desiredgenetic elements (associations) provided therein.

In certain embodiments of the invention, recombination activity,template switching activity, or multiple integration activity ismodulated by altering interaction of the payload with the capsid. In oneembodiment, the lentivirus nucleocapsid (NC) protein is altered bymutating the zinc-finger region so as to disrupt NC-dependentdimerization. See, e.g., Tran et al., 2015, reviewing 5′ UTR and NCfeatures involved in dimerization.

Genetic Perturbations

In one example embodiment, the first polynucleotide may encode one ormore genetic perturbations. The sequences encoding one or more geneticperturbations may comprise an over-expressed gene, siRNAs, microRNAs,regulatory RNAs, ribozymes, antisense RNAs, guide sequences, or asite-specific nuclease. The polynucleotides (e.g., polynucleotides withsequence encoding one or more genetic perturbations) may encode asite-specific nuclease such as, but not limited to, zinc-finger nuclease(ZFN), a transcription activator-like effector nuclease (TALENs) aCRISPR system, a component thereof, a portion thereof, or anycombination thereof. Alternatively or additionally, the polynucleotidesmay encode one or more overexpressed genes, a RNAi based system, acomponent thereof, or a portion thereof. In some examples, thepolynucleotides encode a CRISPR-Cas system or a component thereof. TheCRISPR-Cas system may be a CRISPR-Cas9 system. In some cases, thepolynucleotides encode one or more guide sequences.

Suitable site-specific nuclease systems are described in further detailbelow. The perturbation(s) may comprise single-order perturbations. Theperturbation(s) may comprise combinatorial perturbations. Theperturbations may include gene knock-outs, gene knock-ins,transpositions, inversions, and/or one or more nucleotide insertions,deletions, or substitutions.

In some cases, the polynucleotide comprises a first and a secondengineered associations. The associations may comprise one or moregenetic perturbations. For example, the first engineered association maycomprise a first genetic perturbation and the second engineeredassociation may comprise a second genetic perturbation.

TALENs

In certain embodiments, the sequence encoding the one or more geneticperturbation encodes a (modified) transcription activator-like effectornuclease (TALEN) system. Transcription activator-like effectors (TALEs)can be engineered to bind practically any desired DNA sequence.Exemplary methods of genome editing using the TALEN system can be foundfor example in Cermak T. Doyle E L. Christian M. Wang L. Zhang Y.Schmidt C, et al. Efficient design and assembly of custom TALEN andother TAL effector-based constructs for DNA targeting. Nucleic AcidsRes. 2011; 39:e82; Zhang F. Cong L. Lodato S. Kosuri S. Church G M.Arlotta P Efficient construction of sequence-specific TAL effectors formodulating mammalian transcription. Nat Biotechnol. 2011; 29:149-153 andU.S. Pat. Nos. 8,450,471, 8,440,431 and 8,440,432, all of which arespecifically incorporated by reference. By means of further guidance,and without limitation, naturally occurring TALEs or “wild type TALEs”are nucleic acid binding proteins secreted by numerous species ofproteobacteria. TALE polypeptides contain a nucleic acid binding domaincomposed of tandem repeats of highly conserved monomer polypeptides thatare predominantly 33, 34 or 35 amino acids in length and that differfrom each other mainly in amino acid positions 12 and 13. Inadvantageous embodiments the nucleic acid is DNA. As used herein, theterm “polypeptide monomers”, or “TALE monomers” will be used to refer tothe highly conserved repetitive polypeptide sequences within the TALEnucleic acid binding domain and the term “repeat variable di-residues”or “RVD” will be used to refer to the highly variable amino acids atpositions 12 and 13 of the polypeptide monomers. As provided throughoutthe disclosure, the amino acid residues of the RVD are depicted usingthe IUPAC single letter code for amino acids. A general representationof a TALE monomer which is comprised within the DNA binding domain isX1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates theamino acid position and X represents any amino acid. X12X13 indicate theRVDs. In some polypeptide monomers, the variable amino acid at position13 is missing or absent and in such polypeptide monomers, the RVDconsists of a single amino acid. In such cases the RVD may bealternatively represented as X*, where X represents X12 and (*)indicates that X13 is absent. The DNA binding domain comprises severalrepeats of TALE monomers and this may be represented as(X1-11-(X12X13)-X14-33 or 34 or 35)z, where in an advantageousembodiment, z is at least 5 to 40. In a further advantageous embodiment,z is at least 10 to 26. The TALE monomers have a nucleotide bindingaffinity that is determined by the identity of the amino acids in itsRVD. For example, polypeptide monomers with an RVD of NI preferentiallybind to adenine (A), polypeptide monomers with an RVD of NGpreferentially bind to thymine (T), polypeptide monomers with an RVD ofHD preferentially bind to cytosine (C) and polypeptide monomers with anRVD of NN preferentially bind to both adenine (A) and guanine (G). Inyet another embodiment of the invention, polypeptide monomers with anRVD of IG preferentially bind to T. Thus, the number and order of thepolypeptide monomer repeats in the nucleic acid binding domain of a TALEdetermines its nucleic acid target specificity. In still furtherembodiments of the invention, polypeptide monomers with an RVD of NSrecognize all four base pairs and may bind to A, T, G or C. Thestructure and function of TALEs is further described in, for example,Moscou et al., Science 326:1501 (2009); Boch et al., Science326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153(2011), each of which is incorporated by reference in its entirety. Incertain embodiments, targeting is affected by a polynucleic acid bindingTALEN fragment. In certain embodiments, the targeting domain comprisesor consists of a catalytically inactive TALEN or nucleic acid bindingfragment thereof.

Zn-Finger Nucleases

In certain embodiments, the sequence encoding one or more geneticperturbations comprises or consists of a (modified) zinc-finger nuclease(ZFN) system. The ZFN system uses artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain that can be engineered to target desired DNA sequences. Exemplarymethods of genome editing using ZFNs can be found for example in U.S.Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978,6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626,all of which are specifically incorporated by reference. By means offurther guidance, and without limitation, artificial zinc-finger (ZF)technology involves arrays of ZF modules to target new DNA-binding sitesin the genome. Each finger module in a ZF array targets three DNA bases.A customized array of individual zinc finger domains is assembled into aZF protein (ZFP). ZFPs can comprise a functional domain. The firstsynthetic zinc finger nucleases (ZFNs) were developed by fusing a ZFprotein to the catalytic domain of the Type IIS restriction enzyme FokI.(Kim, Y. G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl.Acad. Sci. U.S.A. 91, 883-887; Kim, Y. G. et al., 1996, Hybridrestriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc.Natl. Acad. Sci. U.S.A. 93, 1156-1160). Increased cleavage specificitycan be attained with decreased off target activity by use of paired ZFNheterodimers, each targeting different nucleotide sequences separated bya short spacer. (Doyon, Y. et al., 2011, Enhancing zinc-finger-nucleaseactivity with improved obligate heterodimeric architectures. Nat.Methods 8, 74-79). ZFPs can also be designed as transcription activatorsand repressors and have been used to target many genes in a wide varietyof organisms. In certain embodiments, the targeting domain comprises orconsists of a nucleic acid binding zinc finger nuclease or a nucleicacid binding fragment thereof. In certain embodiments, the nucleic acidbinding (fragment of) a zinc finger nuclease is catalytically inactive.

Meganuclease

In certain embodiments, the sequences encoding one or more geneticperturbations comprises a (modified) meganuclease, which areendodeoxyribonucleases characterized by a large recognition site(double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methodfor using meganucleases can be found in U.S. Pat. Nos. 8,163,514;8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134,which are specifically incorporated by reference. In certainembodiments, targeting is affected by a polynucleic acid bindingmeganuclease fragment. In certain embodiments, targeting is affected bya polynucleic acid binding catalytically inactive meganuclease(fragment). Accordingly in particular embodiments, the targeting domaincomprises or consists of a nucleic acid binding meganuclease or anucleic acid binding fragment thereof.

CRISPR-Cas Systems

In certain embodiments, the sequence encoding the one or more geneticperturbation encodes a (modified) CRISPR/Cas complex or system. Generalinformation on CRISPR/Cas Systems, components thereof, and delivery ofsuch components, including methods, materials, delivery vehicles,vectors, particles, and making and using thereof, including as toamounts and formulations, as well as CRISPR/Cas-expressing eukaryoticcells, CRISPR/Cas expressing eukaryotes, such as a mouse, is describedherein elsewhere. In certain embodiments, targeting is affected by anoligonucleic acid binding CRISPR protein fragment and/or a gRNA. Incertain embodiments, targeting is affected by a nucleic acid bindingcatalytically inactive CRISPR protein (fragment). Accordingly inparticular embodiments, the targeting domain comprises oligonucleic acidbinding CRISPR protein or an oligonucleic acid binding fragment of aCRISPR protein and/or a gRNA.

As used herein, the term “Cas” generally refers to a (modified) effectorprotein of the CRISPR/Cas system or complex, and can be withoutlimitation a (modified) Cas9, or other enzymes such as Cpf1, C2c1, C2c2,C2c3, group 29, or group 30 protein. The term “Cas” may be used hereininterchangeably with the terms “CRISPR” protein, “CRISPR/Cas protein”,“CRISPR effector”, “CRISPR/Cas effector”, “CRISPR enzyme”, “CRISPR/Casenzyme” and the like, unless otherwise apparent, such as by specific andexclusive reference to Cas9. It is to be understood that the term“CRISPR protein” may be used interchangeably with “CRISPR enzyme”,irrespective of whether the CRISPR protein has altered, such asincreased or decreased (or no) enzymatic activity, compared to the wildtype CRISPR protein. Likewise, as used herein, in certain embodiments,where appropriate and which will be apparent to the skilled person, theterm “nuclease” may refer to a modified nuclease wherein catalyticactivity has been altered, such as having increased or decreasednuclease activity, or no nuclease activity at all, as well as nickaseactivity, as well as otherwise modified nuclease as defined hereinelsewhere, unless otherwise apparent, such as by specific and exclusivereference to unmodified nuclease.

In some embodiments, the CRISPR effector protein is Cas9, Cpf1, C2c1,C2c2, or Cas13a, Cas13b, or Cas13c. In some embodiments, the CRISPReffector protein is a DNA-targeting CRISPR effector protein. In someembodiments, the CRISPR effector protein is a Type-II CRISPR effectorprotein such as Cas9. In some embodiments, the CRISPR effector proteinis a Type-V CRISPR effector protein such as Cpf1 or C2c1. In someembodiments, the CRISPR effector protein is a RNA-targeting CRISPReffector protein. In some embodiments, the CRISPR effector protein is aType-VI CRISPR effector protein such as Cas13a, Cas13b, or Cas13c.

In some embodiments, the CRISPR effector protein is a Cas9, for instanceSaCas9, SpCas9, StCas9, CjCas9 and so forth—any ortholog is envisaged.In some embodiments, the CRISPR effector protein is a Cpf1, for instanceAsCpf1, LbCpf1, FnCpf1 and so forth—any ortholog is envisaged. Incertain embodiments, the targeting component as described hereinaccording to the invention is a (endo)nuclease or a variant thereofhaving altered or modified activity (i.e. a modified nuclease, asdescribed herein elsewhere). In certain embodiments, said nuclease is atargeted or site-specific or homing nuclease or a variant thereof havingaltered or modified activity. In certain embodiments, said nuclease ortargeted/site-specific/homing nuclease is, comprises, consistsessentially of, or consists of a (modified) CRISPR/Cas system orcomplex, a (modified) Cas protein, a (modified) zinc finger, a(modified) zinc finger nuclease (ZFN), a (modified) transcriptionfactor-like effector (TALE), a (modified) transcription factor-likeeffector nuclease (TALEN), or a (modified) meganuclease. In certainembodiments, said (modified) nuclease or targeted/site-specific/homingnuclease is, comprises, consists essentially of, or consists of a(modified) RNA-guided nuclease.

In particular embodiments, more particularly where the nuclease is aCRISPR protein, the targeting domain further comprises a guide moleculewhich targets a selected nucleic acid. For instance, in the context ofthe CRISPR/Cas system, the guide RNA is capable of hybridizing with aselected nucleic acid sequence. As used herein, “hybridization” or“hybridizing” refers to a reaction in which one or more polynucleotidesreact to form a complex that is stabilized via hydrogen bonding betweenthe bases of the nucleotide residues. The hydrogen bonding may occur byWatson Crick base pairing, Hoogsteen binding, or in any other sequencespecific manner. The complex may comprise two strands forming a duplexstructure, three or more strands forming a multi stranded complex, asingle self-hybridizing strand, or any combination of these. Ahybridization reaction may constitute a step in a more extensiveprocess, such as the initiation of PGR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

Guide Sequences

In certain example embodiments, one of the engineered associations maycomprise one of the above Cas proteins. In another embodiment, one ofthe engineered associations may comprise a Cas protein and secondengineered association may comprise a guide sequence. In yet anotherembodiment, the engineered associations may comprise two or more guidesequences. As used herein, the term “guide sequence” and “guidemolecule” in the context of a CRISPR-Cas system, comprises anypolynucleotide sequence having sufficient complementarity with a targetnucleic acid sequence to hybridize with the target nucleic acid sequenceand direct sequence-specific binding of a nucleic acid-targeting complexto the target nucleic acid sequence. The guide sequences made using themethods disclosed herein may be a full-length guide sequence, atruncated guide sequence, a full-length sgRNA sequence, a truncatedsgRNA sequence, or an E+F sgRNA sequence. In some embodiments, thedegree of complementarity of the guide sequence to a given targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. In certain example embodiments, the guide moleculecomprises a guide sequence that may be designed to have at least onemismatch with the target sequence, such that a RNA duplex formed betweenthe guide sequence and the target sequence. Accordingly, the degree ofcomplementarity is preferably less than 99%. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less. In particular embodiments, theguide sequence is designed to have a stretch of two or more adjacentmismatching nucleotides, such that the degree of complementarity overthe entire guide sequence is further reduced. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less, more particularly, about 92% orless, more particularly about 88% or less, more particularly about 84%or less, more particularly about 80% or less, more particularly about76% or less, more particularly about 72% or less, depending on whetherthe stretch of two or more mismatching nucleotides encompasses 2, 3, 4,5, 6 or 7 nucleotides, etc. In some embodiments, aside from the stretchof one or more mismatching nucleotides, the degree of complementarity,when optimally aligned using a suitable alignment algorithm, is about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within a nucleicacid-targeting guide RNA) to direct sequence-specific binding of anucleic acid-targeting complex to a target nucleic acid sequence may beassessed by any suitable assay. For example, the components of a nucleicacid-targeting CRISPR system sufficient to form a nucleic acid-targetingcomplex, including the guide sequence to be tested, may be provided to ahost cell having the corresponding target nucleic acid sequence, such asby transfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence (or a sequence in the vicinity thereof) maybe evaluated in a test tube by providing the target nucleic acidsequence, components of a nucleic acid-targeting complex, including theguide sequence to be tested and a control guide sequence different fromthe test guide sequence, and comparing binding or rate of cleavage at orin the vicinity of the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art. A guide sequence, and hence a nucleicacid-targeting guide RNA may be selected to target any target nucleicacid sequence.

In certain embodiments, the guide sequence or spacer length of the guidemolecules is from 15 to 50 nt. In certain embodiments, the spacer lengthof the guide RNA is at least 15 nucleotides. In certain embodiments, thespacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23,or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt,e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt,from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.In certain example embodiments, the guide sequence is 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39 40, 41, 42, 43, 44, 45, 46, 47 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nt.

In some embodiments, the guide sequence is an RNA sequence of between 10to 50 nt in length, but more particularly of about 20-30 ntadvantageously about 20 nt, 23-25 nt or 24 nt. The guide sequence isselected so as to ensure that it hybridizes to the target sequence. Thisis described more in detail below. Selection can encompass further stepswhich increase efficacy and specificity.

In some embodiments, the guide sequence has a canonical length (e.g.,about 15-30 nt) is used to hybridize with the target RNA or DNA. In someembodiments, a guide molecule is longer than the canonical length(e.g., >30 nt) is used to hybridize with the target RNA or DNA, suchthat a region of the guide sequence hybridizes with a region of the RNAor DNA strand outside of the Cas-guide target complex. This can be ofinterest where additional modifications, such deamination of nucleotidesis of interest. In alternative embodiments, it is of interest tomaintain the limitation of the canonical guide sequence length.

In some embodiments, the sequence of the guide molecule (direct repeatand/or spacer) is selected to reduce the degree secondary structurewithin the guide molecule. In some embodiments, about or less than about75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of thenucleotides of the nucleic acid-targeting guide RNA participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g., A. R. Gruber et al., 2008,Cell 106(1): 23-24; and P A Carr and G M Church, 2009, NatureBiotechnology 27(12): 1151-62).

In some embodiments, it is of interest to reduce the susceptibility ofthe guide molecule to RNA cleavage, such as to cleavage by Cas13.Accordingly, in particular embodiments, the guide molecule is adjustedto avoid cleavage by Cas13 or other RNA-cleaving enzymes.

In certain embodiments, the guide molecule comprises non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications. Preferably,these non-naturally occurring nucleic acids and non-naturally occurringnucleotides are located outside the guide sequence. Non-naturallyoccurring nucleic acids can include, for example, mixtures of naturallyand non-naturally occurring nucleotides. Non-naturally occurringnucleotides and/or nucleotide analogs may be modified at the ribose,phosphate, and/or base moiety. In an embodiment of the invention, aguide nucleic acid comprises ribonucleotides and non-ribonucleotides. Inone such embodiment, a guide comprises one or more ribonucleotides andone or more deoxyribonucleotides. In an embodiment of the invention, theguide comprises one or more non-naturally occurring nucleotide ornucleotide analog such as a nucleotide with phosphorothioate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, or 2′-fluoro analogs. Further examples ofmodified bases include, but are not limited to, 2-aminopurine,5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS),S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guides can compriseincreased stability and increased activity as compared to unmodifiedguides, though on-target vs. off-target specificity is not predictable.(See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015 Ragdarm et al., 0215, PNAS, E7110-E7111;Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front.Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma etal., MedChemComm., 2014, 5:1454-1471; Hendel et al., Nat. Biotechnol.(2015) 33(9): 985-989; Li et al., Nature Biomedical Engineering, 2017,1, 0066 DOI:10.1038/s41551-017-0066). In some embodiments, the 5′ and/or3′ end of a guide RNA is modified by a variety of functional moietiesincluding fluorescent dyes, polyethylene glycol, cholesterol, proteins,or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). Incertain embodiments, a guide comprises ribonucleotides in a region thatbinds to a target RNA and one or more deoxyribonucleotides and/ornucleotide analogs in a region that binds to Cas13. In an embodiment ofthe invention, deoxyribonucleotides and/or nucleotide analogs areincorporated in engineered guide structures, such as, withoutlimitation, stem-loop regions, and the seed region. For Cas13 guide, incertain embodiments, the modification is not in the 5′-handle of thestem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemically modified with 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP). Such modification can enhance genome editing efficiency(see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). In certainembodiments, all of the phosphodiester bonds of a guide are substitutedwith phosphorothioates (PS) for enhancing levels of gene disruption. Incertain embodiments, more than five nucleotides at the 5′ and/or the 3′end of the guide are chemically modified with 2′-O-Me, 2′-F orS-constrained ethyl(cEt). Such chemically modified guide can mediateenhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS,E7110-E7111). In an embodiment of the invention, a guide is modified tocomprise a chemical moiety at its 3′ and/or 5′ end. Such moietiesinclude, but are not limited to amine, azide, alkyne, thio,dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, thechemical moiety is conjugated to the guide by a linker, such as an alkylchain. In certain embodiments, the chemical moiety of the modified guidecan be used to attach the guide to another molecule, such as DNA, RNA,protein, or nanoparticles. Such chemically modified guide can be used toidentify or enrich cells genetically edited by a CRISPR system (see Leeet al., eLife, 2017, 6:e25312, DOI:10.7554).

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (T), N1-methylpseudouridine (me1Ψ),5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2′-O-methyl3′phosphorothioate (MS), S-constrained ethyl(cEt), phosphorothioate(PS), or 2′-O-methyl 3′thioPACE (MSP). In some embodiments, the guidecomprises one or more of phosphorothioate modifications. In certainembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemicallymodified. In certain embodiments, one or more nucleotides in the seedregion are chemically modified. In certain embodiments, one or morenucleotides in the 3′-terminus are chemically modified. In certainembodiments, none of the nucleotides in the 5′-handle is chemicallymodified. In some embodiments, the chemical modification in the seedregion is a minor modification, such as incorporation of a 2′-fluoroanalog. In a specific embodiment, one nucleotide of the seed region isreplaced with a 2′-fluoro analog. In some embodiments, 5 to 10nucleotides in the 3′-terminus are chemically modified. Such chemicalmodifications at the 3′-terminus of the Cas13 CrRNA may improve Cas13activity. In a specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides in the 3′-terminus are replaced with 2′-fluoro analogues. Ina specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides inthe 3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the modified loop comprises 3, 4, or 5 nucleotides.In certain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

In some embodiments, the guide molecule forms a stemloop with a separatenon-covalently linked sequence, which can be DNA or RNA. In particularembodiments, the sequences forming the guide are first synthesized usingthe standard phosphoramidite synthetic protocol (Herdewijn, P., ed.,Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methodsand Applications, Humana Press, New Jersey (2012)). In some embodiments,these sequences can be functionalized to contain an appropriatefunctional group for ligation using the standard protocol known in theart (Hermanson, G. T., Bioconjugate Techniques, Academic Press (2013)).Examples of functional groups include, but are not limited to, hydroxyl,amine, carboxylic acid, carboxylic acid halide, carboxylic acid activeester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl,hydrozide, semicarbazide, thio semicarbazide, thiol, maleimide,haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide. Once thissequence is functionalized, a covalent chemical bond or linkage can beformed between this sequence and the direct repeat sequence. Examples ofchemical bonds include, but are not limited to, those based oncarbamates, ethers, esters, amides, imines, amidines, aminotrizines,hydrozone, disulfides, thioethers, thioesters, phosphorothioates,phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides,ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—Cbond forming groups such as Diels-Alder cyclo-addition pairs orring-closing metathesis pairs, and Michael reaction pairs.

In some embodiments, these stem-loop forming sequences can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In certain embodiments, the guide molecule comprises (1) a guidesequence capable of hybridizing to a target locus and (2) a tracr mateor direct repeat sequence whereby the direct repeat sequence is locatedupstream (i.e., 5′) from the guide sequence. In a particular embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of th guidesequence is approximately within the first 10 nucleotides of the guidesequence.

In a particular embodiment the guide molecule comprises a guide sequencelinked to a direct repeat sequence, wherein the direct repeat sequencecomprises one or more stem loops or optimized secondary structures. Inparticular embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loops or optimized secondary structures. In particularembodiments the guide molecule comprises or consists of the guidesequence linked to all or part of the natural direct repeat sequence. Atypical Type V or Type VI CRISPR-cas guide molecule comprises (in 3′ to5′ direction or in 5′ to 3′ direction): a guide sequence a firstcomplimentary stretch (the “repeat”), a loop (which is typically 4 or 5nucleotides long), a second complementary stretch (the “anti-repeat”being complementary to the repeat), and a poly A (often poly U in RNA)tail (terminator). In certain embodiments, the direct repeat sequenceretains its natural architecture and forms a single stem loop. Inparticular embodiments, certain aspects of the guide architecture can bemodified, for example by addition, subtraction, or substitution offeatures, whereas certain other aspects of guide architecture aremaintained. Preferred locations for engineered guide moleculemodifications, including but not limited to insertions, deletions, andsubstitutions include guide termini and regions of the guide moleculethat are exposed when complexed with the CRISPR-Cas protein and/ortarget, for example the stemloop of the direct repeat sequence.

In particular embodiments, the stem comprises at least about 4 bpcomprising complementary X and Y sequences, although stems of more,e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs arealso contemplated. Thus, for example X2-10 and Y2-10 (wherein X and Yrepresent any complementary set of nucleotides) may be contemplated. Inone aspect, the stem made of the X and Y nucleotides, together with theloop will form a complete hairpin in the overall secondary structure;and, this may be advantageous and the amount of base pairs can be anyamount that forms a complete hairpin. In one aspect, any complementaryX:Y basepairing sequence (e.g., as to length) is tolerated, so long asthe secondary structure of the entire guide molecule is preserved. Inone aspect, the loop that connects the stem made of X:Y basepairs can beany sequence of the same length (e.g., 4 or 5 nucleotides) or longerthat does not interrupt the overall secondary structure of the guidemolecule. In one aspect, the stemloop can further comprise, e.g. an MS2aptamer. In one aspect, the stem comprises about 5-7 bp comprisingcomplementary X and Y sequences, although stems of more or fewerbasepairs are also contemplated. In one aspect, non-Watson Crickbasepairing is contemplated, where such pairing otherwise generallypreserves the architecture of the stemloop at that position.

In particular embodiments the natural hairpin or stemloop structure ofthe guide molecule is extended or replaced by an extended stemloop. Ithas been demonstrated that extension of the stem can enhance theassembly of the guide molecule with the CRISPR-Cas proten (Chen et al.Cell. (2013); 155(7): 1479-1491). In particular embodiments the stem ofthe stemloop is extended by at least 1, 2, 3, 4, 5 or more complementarybasepairs (i.e. corresponding to the addition of 2, 4, 6, 8, 10 or morenucleotides in the guide molecule). In particular embodiments these arelocated at the end of the stem, adjacent to the loop of the stemloop.

In particular embodiments, the susceptibility of the guide molecule toRNAses or to decreased expression can be reduced by slight modificationsof the sequence of the guide molecule which do not affect its function.For instance, in particular embodiments, premature termination oftranscription, such as premature transcription of U6 Pol-III, can beremoved by modifying a putative Pol-III terminator (4 consecutive U's)in the guide molecules sequence. Where such sequence modification isrequired in the stemloop of the guide molecule, it is preferably ensuredby a basepair flip.

In a particular embodiment the direct repeat may be modified to compriseone or more protein-binding RNA aptamers. In a particular embodiment,one or more aptamers may be included such as part of optimized secondarystructure. Such aptamers may be capable of binding a bacteriophage coatprotein as detailed further herein.

In some embodiments, the guide molecule forms a duplex with a target RNAcomprising at least one target cytosine residue to be edited. Uponhybridization of the guide RNA molecule to the target RNA, the cytidinedeaminase binds to the single strand RNA in the duplex made accessibleby the mismatch in the guide sequence and catalyzes deamination of oneor more target cytosine residues comprised within the stretch ofmismatching nucleotides.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. The target sequencemay be mRNA.

In certain embodiments, the target sequence should be associated with aPAM (protospacer adjacent motif) or PFS (protospacer flanking sequenceor site); that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments of the present inventionwhere the CRISPR-Cas protein is a Cas13 protein, the complementarysequence of the target sequence is downstream or 3′ of the PAM orupstream or 5′ of the PAM. The precise sequence and length requirementsfor the PAM differ depending on the Cas13 protein used, but PAMs aretypically 2-5 base pair sequences adjacent the protospacer (that is, thetarget sequence). Examples of the natural PAM sequences for differentCas13 orthologues are provided herein below and the skilled person willbe able to identify further PAM sequences for use with a given Cas13protein.

Further, engineering of the PAM Interacting (PI) domain may allowprograming of PAM specificity, improve target site recognition fidelity,and increase the versatility of the CRISPR-Cas protein, for example asdescribed for Cas9 in Kleinstiver B P et al. Engineered CRISPR-Cas9nucleases with altered PAM specificities. Nature. 2015 Jul. 23;523(7561):481-5. doi: 10.1038/nature14592. As further detailed herein,the skilled person will understand that Cas13 proteins may be modifiedanalogously.

In particular embodiment, the guide is an escorted guide. By “escorted”is meant that the CRISPR-Cas system or complex or guide is delivered toa selected time or place within a cell, so that activity of theCRISPR-Cas system or complex or guide is spatially or temporallycontrolled. For example, the activity and destination of the 3CRISPR-Cas system or complex or guide may be controlled by an escort RNAaptamer sequence that has binding affinity for an aptamer ligand, suchas a cell surface protein or other localized cellular component.Alternatively, the escort aptamer may for example be responsive to anaptamer effector on or in the cell, such as a transient effector, suchas an external energy source that is applied to the cell at a particulartime.

The escorted CRISPR-Cas systems or complexes may have a guide moleculewith a functional structure designed to improve guide moleculestructure, architecture, stability, genetic expression, or anycombination thereof. Such a structure can include an aptamer.

Aptamers are biomolecules that can be designed or selected to bindtightly to other ligands, for example using a technique calledsystematic evolution of ligands by exponential enrichment (SELEX; TuerkC, Gold L: “Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990,249:505-510). Nucleic acid aptamers can for example be selected frompools of random-sequence oligonucleotides, with high binding affinitiesand specificities for a wide range of biomedically relevant targets,suggesting a wide range of therapeutic utilities for aptamers (Keefe,Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers astherapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). Thesecharacteristics also suggest a wide range of uses for aptamers as drugdelivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology andaptamers: applications in drug delivery.” Trends in biotechnology 26.8(2008): 442-449; and, Hicke B J, Stephens A W. “Escort aptamers: adelivery service for diagnosis and therapy.” J Clin Invest 2000,106:923-928.). Aptamers may also be constructed that function asmolecular switches, responding to a que by changing properties, such asRNA aptamers that bind fluorophores to mimic the activity of greenfluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R.Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042(2011): 642-646). It has also been suggested that aptamers may be usedas components of targeted siRNA therapeutic delivery systems, forexample targeting cell surface proteins (Zhou, Jiehua, and John J.Rossi. “Aptamer-targeted cell-specific RNA interference.” Silence 1.1(2010): 4).

Accordingly, in particular embodiments, the guide molecule is modified,e.g., by one or more aptamer(s) designed to improve guide moleculedelivery, including delivery across the cellular membrane, tointracellular compartments, or into the nucleus. Such a structure caninclude, either in addition to the one or more aptamer(s) or withoutsuch one or more aptamer(s), moiety(ies) so as to render the guidemolecule deliverable, inducible or responsive to a selected effector.The invention accordingly comprehends an guide molecule that responds tonormal or pathological physiological conditions, including withoutlimitation pH, hypoxia, 02 concentration, temperature, proteinconcentration, enzymatic concentration, lipid structure, light exposure,mechanical disruption (e.g. ultrasound waves), magnetic fields, electricfields, or electromagnetic radiation.

Light responsiveness of an inducible system may be achieved via theactivation and binding of cryptochrome-2 and CIB 1. Blue lightstimulation induces an activating conformational change incryptochrome-2, resulting in recruitment of its binding partner CIB1.This binding is fast and reversible, achieving saturation in <15 secfollowing pulsed stimulation and returning to baseline<15 min after theend of stimulation. These rapid binding kinetics result in a systemtemporally bound only by the speed of transcription/translation andtranscript/protein degradation, rather than uptake and clearance ofinducing agents. Cryptochrome-2 activation is also highly sensitive,allowing for the use of low light intensity stimulation and mitigatingthe risks of phototoxicity. Further, in a context such as the intactmammalian brain, variable light intensity may be used to control thesize of a stimulated region, allowing for greater precision than vectordelivery alone may offer.

The disclosure contemplates energy sources such as electromagneticradiation, sound energy or thermal energy to induce the guide.Advantageously, the electromagnetic radiation is a component of visiblelight. In a preferred embodiment, the light is a blue light with awavelength of about 450 to about 495 nm. In an especially preferredembodiment, the wavelength is about 488 nm. In another preferredembodiment, the light stimulation is via pulses. The light power mayrange from about 0-9 mW/cm². In a preferred embodiment, a stimulationparadigm of as low as 0.25 sec every 15 sec should result in maximalactivation.

The chemical or energy sensitive guide may undergo a conformationalchange upon induction by the binding of a chemical source or by theenergy allowing it act as a guide and have the Cas13 CRISPR-Cas systemor complex function. The invention can involve applying the chemicalsource or energy so as to have the guide function and the Cas13CRISPR-Cas system or complex function; and optionally furtherdetermining that the expression of the genomic locus is altered.

There are several different designs of this chemical induciblesystem: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g., stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g.,www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAIbased system inducible by Gibberellin (GA) (see, e.g.,www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

A chemical inducible system can be an estrogen receptor (ER) basedsystem inducible by 4-hydroxytamoxifen (4OHT) (see, e.g.,//www.pnas.org/content/104/3/1027.abstract). A mutated ligand-bindingdomain of the estrogen receptor called ERT2 translocates into thenucleus of cells upon binding of 4-hydroxytamoxifen. In furtherembodiments of the invention any naturally occurring or engineeredderivative of any nuclear receptor, thyroid hormone receptor, retinoicacid receptor, estrogen receptor, estrogen-related receptor,glucocorticoid receptor, progesterone receptor, androgen receptor may beused in inducible systems analogous to the ER based inducible system.

Another example inducible system is based on the design using Transientreceptor potential (TRP) ion channel based system inducible by energy,heat or radio-wave (see, e.g., www.sciencemag.org/content/336/6081/604).These TRP family proteins respond to different stimuli, including lightand heat. When this protein is activated by light or heat, the ionchannel will open and allow the entering of ions such as calcium intothe plasma membrane. This influx of ions will bind to intracellular ioninteracting partners linked to a polypeptide including the guide and theother components of the Cas13 CRISPR-Cas complex or system, and thebinding will induce the change of sub-cellular localization of thepolypeptide, leading to the entire polypeptide entering the nucleus ofcells. Once inside the nucleus, the guide protein and the othercomponents of the Cas13 CRISPR-Cas complex will be active and modulatingtarget gene expression in cells.

While light activation may be an advantageous embodiment, sometimes itmay be disadvantageous especially for in vivo applications in which thelight may not penetrate the skin or other organs. In this instance,other methods of energy activation are contemplated, in particular,electric field energy and/or ultrasound which have a similar effect.

Electric field energy is preferably administered substantially asdescribed in the art, using one or more electric pulses of from about 1Volt/cm to about 10 kVolts/cm under in vivo conditions. Instead of or inaddition to the pulses, the electric field may be delivered in acontinuous manner. The electric pulse may be applied for between 1 μsand 500 milliseconds, preferably between 1 μs and 100 milliseconds. Theelectric field may be applied continuously or in a pulsed manner for 5about minutes.

As used herein, ‘electric field energy’ is the electrical energy towhich a cell is exposed. Preferably the electric field has a strength offrom about 1 Volt/cm to about 10 kVolts/cm or more under in vivoconditions (see WO97/49450).

As used herein, the term “electric field” includes one or more pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave and/or modulated square wave forms.References to electric fields and electricity should be taken to includereference the presence of an electric potential difference in theenvironment of a cell. Such an environment may be set up by way ofstatic electricity, alternating current (AC), direct current (DC), etc.,as known in the art. The electric field may be uniform, non-uniform orotherwise, and may vary in strength and/or direction in a time dependentmanner.

Single or multiple applications of electric field, as well as single ormultiple applications of ultrasound are also possible, in any order andin any combination. The ultrasound and/or the electric field may bedelivered as single or multiple continuous applications, or as pulses(pulsatile delivery).

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the agent ofinterest and placed between electrodes such as parallel plates. Then,the electrodes apply an electrical field to the cell/implant mixture.Examples of systems that perform in vitro electroporation include theElectro Cell Manipulator ECM600 product, and the Electro Square PoratorT820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat.No. 5,869,326).

The known electroporation techniques (both in vitro and in vivo)function by applying a brief high voltage pulse to electrodes positionedaround the treatment region. The electric field generated between theelectrodes causes the cell membranes to temporarily become porous,whereupon molecules of the agent of interest enter the cells. In knownelectroporation applications, this electric field comprises a singlesquare wave pulse on the order of 1000 V/cm, of about 100 .mu.sduration. Such a pulse may be generated, for example, in knownapplications of the Electro Square Porator T820.

Preferably, the electric field has a strength of from about 1 V/cm toabout 10 kV/cm under in vitro conditions. Thus, the electric field mayhave a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. Morepreferably from about 0.5 kV/cm to about 4.0 kV/cm under in vitroconditions. Preferably the electric field has a strength of from about 1V/cm to about 10 kV/cm under in vivo conditions. However, the electricfield strengths may be lowered where the number of pulses delivered tothe target site are increased. Thus, pulsatile delivery of electricfields at lower field strengths is envisaged.

Preferably the application of the electric field is in the form ofmultiple pulses such as double pulses of the same strength andcapacitance or sequential pulses of varying strength and/or capacitance.As used herein, the term “pulse” includes one or more electric pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave/square wave forms.

Preferably the electric pulse is delivered as a waveform selected froman exponential wave form, a square wave form, a modulated wave form anda modulated square wave form.

A preferred embodiment employs direct current at low voltage. Thus,Applicants disclose the use of an electric field which is applied to thecell, tissue or tissue mass at a field strength of between 1V/cm and20V/cm, for a period of 100 milliseconds or more, preferably 15 minutesor more.

Ultrasound is advantageously administered at a power level of from about0.05 W/cm² to about 100 W/cm². Diagnostic or therapeutic ultrasound maybe used, or combinations thereof.

As used herein, the term “ultrasound” refers to a form of energy whichconsists of mechanical vibrations the frequencies of which are so highthey are above the range of human hearing. Lower frequency limit of theultrasonic spectrum may generally be taken as about 20 kHz. Mostdiagnostic applications of ultrasound employ frequencies in the range 1and 15 MHz′ (From Ultrasonics in Clinical Diagnosis, P. N. T. Wells,ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY,1977]).

Ultrasound has been used in both diagnostic and therapeuticapplications. When used as a diagnostic tool (“diagnostic ultrasound”),ultrasound is typically used in an energy density range of up to about100 mW/cm² (FDA recommendation), although energy densities of up to 750mW/cm² have been used. In physiotherapy, ultrasound is typically used asan energy source in a range up to about 3 to 4 W/cm² (WHOrecommendation). In other therapeutic applications, higher intensitiesof ultrasound may be employed, for example, HIFU at 100 W/cm up to 1kW/cm² (or even higher) for short periods of time. The term “ultrasound”as used in this specification is intended to encompass diagnostic,therapeutic and focused ultrasound.

Focused ultrasound (FUS) allows thermal energy to be delivered withoutan invasive probe (see Morocz et al 1998 Journal of Magnetic ResonanceImaging Vol. 8, No. 1, pp. 136-142). Another form of focused ultrasoundis high intensity focused ultrasound (HIFU) which is reviewed byMoussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 andTranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106.

Preferably, a combination of diagnostic ultrasound and a therapeuticultrasound is employed. This combination is not intended to be limiting,however, and the skilled reader will appreciate that any variety ofcombinations of ultrasound may be used. Additionally, the energydensity, frequency of ultrasound, and period of exposure may be varied.

Preferably the exposure to an ultrasound energy source is at a powerdensity of from about 0.05 to about 100 Wcm⁻². Even more preferably, theexposure to an ultrasound energy source is at a power density of fromabout 1 to about 15 Wcm⁻².

Preferably the exposure to an ultrasound energy source is at a frequencyof from about 0.015 to about 10.0 MHz. More preferably the exposure toan ultrasound energy source is at a frequency of from about 0.02 toabout 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound isapplied at a frequency of 3 MHz.

Preferably the exposure is for periods of from about 10 milliseconds toabout 60 minutes. Preferably the exposure is for periods of from about 1second to about 5 minutes. More preferably, the ultrasound is appliedfor about 2 minutes. Depending on the particular target cell to bedisrupted, however, the exposure may be for a longer duration, forexample, for 15 minutes.

Advantageously, the target tissue is exposed to an ultrasound energysource at an acoustic power density of from about 0.05 Wcm⁻² to about 10Wcm⁻² with a frequency ranging from about 0.015 to about 10 MHz (see WO98/52609). However, alternatives are also possible, for example,exposure to an ultrasound energy source at an acoustic power density ofabove 100 Wcm⁻², but for reduced periods of time, for example, 1000Wcm⁻² for periods in the millisecond range or less.

Preferably the application of the ultrasound is in the form of multiplepulses; thus, both continuous wave and pulsed wave (pulsatile deliveryof ultrasound) may be employed in any combination. For example,continuous wave ultrasound may be applied, followed by pulsed waveultrasound, or vice versa. This may be repeated any number of times, inany order and combination. The pulsed wave ultrasound may be appliedagainst a background of continuous wave ultrasound, and any number ofpulses may be used in any number of groups.

Preferably, the ultrasound may comprise pulsed wave ultrasound. In ahighly preferred embodiment, the ultrasound is applied at a powerdensity of 0.7 Wcm-2 or 1.25 Wcm-2 as a continuous wave. Higher powerdensities may be employed if pulsed wave ultrasound is used.

Use of ultrasound is advantageous as, like light, it may be focusedaccurately on a target. Moreover, ultrasound is advantageous as it maybe focused more deeply into tissues unlike light. It is therefore bettersuited to whole-tissue penetration (such as but not limited to a lobe ofthe liver) or whole organ (such as but not limited to the entire liveror an entire muscle, such as the heart) therapy. Another importantadvantage is that ultrasound is a non-invasive stimulus which is used ina wide variety of diagnostic and therapeutic applications. By way ofexample, ultrasound is well known in medical imaging techniques and,additionally, in orthopedic therapy. Furthermore, instruments suitablefor the application of ultrasound to a subject vertebrate are widelyavailable and their use is well known in the art.

In particular embodiments, the guide molecule is modified by a secondarystructure to increase the specificity of the CRISPR-Cas system and thesecondary structure can protect against exonuclease activity and allowfor 5′ additions to the guide sequence also referred to herein as aprotected guide molecule.

In one aspect, the invention provides for hybridizing a “protector RNA”to a sequence of the guide molecule, wherein the “protector RNA” is anRNA strand complementary to the 3′ end of the guide molecule to therebygenerate a partially double-stranded guide RNA. In an embodiment of theinvention, protecting mismatched bases (i.e. the bases of the guidemolecule which do not form part of the guide sequence) with a perfectlycomplementary protector sequence decreases the likelihood of target RNAbinding to the mismatched basepairs at the 3′ end. In particularembodiments of the invention, additional sequences comprising anextended length may also be present within the guide molecule such thatthe guide comprises a protector sequence within the guide molecule. This“protector sequence” ensures that the guide molecule comprises a“protected sequence” in addition to an “exposed sequence” (comprisingthe part of the guide sequence hybridizing to the target sequence). Inparticular embodiments, the guide molecule is modified by the presenceof the protector guide to comprise a secondary structure such as ahairpin. Advantageously there are three or four to thirty or more, e.g.,about 10 or more, contiguous base pairs having complementarity to theprotected sequence, the guide sequence or both. It is advantageous thatthe protected portion does not impede thermodynamics of the CRISPR-Cassystem interacting with its target. By providing such an extensionincluding a partially double stranded guide molecule, the guide moleculeis considered protected and results in improved specific binding of theCRISPR-Cas complex, while maintaining specific activity.

In particular embodiments, use is made of a truncated guide (tru-guide),i.e. a guide molecule which comprises a guide sequence which istruncated in length with respect to the canonical guide sequence length.As described by Nowak et al. (Nucleic Acids Res (2016) 44 (20):9555-9564), such guides may allow catalytically active CRISPR-Cas enzymeto bind its target without cleaving the target RNA. In particularembodiments, a truncated guide is used which allows the binding of thetarget but retains only nickase activity of the CRISPR-Cas enzyme.

In certain example embodiments, the system may comprise a first guidesequence and a second guide sequence such as used in paired nickasesystem or self-inactivating systems. Paired nickase systems are used,for example, to minimize off-target effects. Typically, guides aredesigned in pairs and used with a nickase to introduce two nicks, one oneach strand, into a DNA duplex, each nick targeted to adjacent butdifferent sequences of a genomic locus. In an embodiment, the guides areexpressed from the same promoter. In and embodiment, the guides are intandem. In such embodiments, the guides are designed to work together,encoded on a single polynucleotide and packaged together. By reducing oreliminating recombination or template switching activity, the inventionimproves the performance of multiplexed nickase systems comprising twoor more guide pairs (i.e., targeting two or more genetic loci). In aself-inactivating (SIN) system two or more loci are targeted. One targetcomprises, for example, a genomic locus intended to be modified and thesecond target comprises a locus associated with a CRISPR systemcomponent whereby the function of the CRISPR system may be targeted. Incertain SIN systems, it will be desired to maintain the linkage of aguide that targets the genomic locus with the guide that targets theCRISPR component. Example self-inactivating systems are disclosed inWO/2015/070083, WO/2015/089354, and WO/2015/089351. Example tandem guidesystems are disclosed in WO/2014/204724, WO/2014/093622, andWO/2014/204725.

Unique Molecular Sequence

In certain example embodiments, one of the engineered association may aunique molecular identifier. The unique molecular identifier may be arandom nucleotide sequence that uniquely identifies the polynucleotideand/or the other engineered associations encoded on the firstpolynucleotide. In certain example embodiment, the unique molecularsequence may be a barcode. The term “barcode” as used herein refers to ashort sequence of nucleotides (for example, DNA or RNA) that is used asan identifier for an associated molecule, such as a target moleculeand/or target nucleic acid, or as an identifier of the source of anassociated molecule, such as a cell-of-origin. A barcode may also referto any unique, non-naturally occurring, nucleic acid sequence that maybe used to identify the originating source of a nucleic acid fragment.Although it is not necessary to understand the mechanism of aninvention, it is believed that the barcode sequence provides ahigh-quality individual read of a barcode associated with a single cell,a viral vector, labeling ligand (e.g., an aptamer), protein, shRNA,sgRNA or cDNA such that multiple species can be sequenced together.

Barcoding may be performed based on any of the compositions or methodsdisclosed in patent publication WO 2014047561 A1, Compositions andmethods for labeling of agents, incorporated herein in its entirety. Incertain embodiments barcoding uses an error correcting scheme (T. K.Moon, Error Correction Coding: Mathematical Methods and Algorithms(Wiley, New York, ed. 1, 2005)). Not being bound by a theory, amplifiedsequences from single cells can be sequenced together and resolved basedon the barcode associated with each cell.

In preferred embodiments, sequencing is performed using unique molecularidentifiers (UMI). The term “unique molecular identifiers” (UMI) as usedherein refers to a sequencing linker or a subtype of nucleic acidbarcode used in a method that uses molecular tags to detect and quantifyunique amplified products. A UMI is used to distinguish effects througha single clone from multiple clones. The term “clone” as used herein mayrefer to a single mRNA or target nucleic acid to be sequenced. The UMImay also be used to determine the number of transcripts that gave riseto an amplified product, or in the case of target barcodes as describedherein, the number of binding events. In preferred embodiments, theamplification is by PCR or multiple displacement amplification (MDA).

In certain embodiments, an UMI with a random sequence of between 4 and20 base pairs is added to a template, which is amplified and sequenced.In preferred embodiments, the UMI is added to the 5′ end of thetemplate. Sequencing allows for high resolution reads, enabling accuratedetection of true variants. As used herein, a “true variant” will bepresent in every amplified product originating from the original cloneas identified by aligning all products with a UMI. Each clone amplifiedwill have a different random UMI that will indicate that the amplifiedproduct originated from that clone. Background caused by the fidelity ofthe amplification process can be eliminated because true variants willbe present in all amplified products and background representing randomerror will only be present in single amplification products (See e.g.,Islam S. et al., 2014. Nature Methods No:11, 163-166). Not being boundby a theory, the UMI's are designed such that assignment to the originalcan take place despite up to 4-7 errors during amplification orsequencing. Not being bound by a theory, an UMI may be used todiscriminate between true barcode sequences.

Unique molecular identifiers can be used, for example, to normalizesamples for variable amplification efficiency. For example, in variousembodiments, featuring a solid or semisolid support (for example ahydrogel bead), to which nucleic acid barcodes (for example a pluralityof barcodes sharing the same sequence) are attached, each of thebarcodes may be further coupled to a unique molecular identifier, suchthat every barcode on the particular solid or semisolid support receivesa distinct unique molecule identifier. A unique molecular identifier canthen be, for example, transferred to a target molecule with theassociated barcode, such that the target molecule receives not only anucleic acid barcode, but also an identifier unique among theidentifiers originating from that solid or semisolid support.

A nucleic acid barcode or UMI can have a length of at least, forexample, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90,or 100 nucleotides, and can be in single- or double-stranded form.Target molecule and/or target nucleic acids can be labeled with multiplenucleic acid barcodes in combinatorial fashion, such as a nucleic acidbarcode concatemer. Typically, a nucleic acid barcode is used toidentify a target molecule and/or target nucleic acid as being from aparticular discrete volume, having a particular physical property (forexample, affinity, length, sequence, etc.), or having been subject tocertain treatment conditions. Target molecule and/or target nucleic acidcan be associated with multiple nucleic acid barcodes to provideinformation about all of these features (and more). Each member of agiven population of UMIs, on the other hand, is typically associatedwith (for example, covalently bound to or a component of the samemolecule as) individual members of a particular set of identical,specific (for example, discreet volume-, physical property-, ortreatment condition-specific) nucleic acid barcodes. Thus, for example,each member of a set of origin-specific nucleic acid barcodes, or othernucleic acid identifier or connector oligonucleotide, having identicalor matched barcode sequences, may be associated with (for example,covalently bound to or a component of the same molecule as) a distinctor different UMI.

As disclosed herein, unique nucleic acid identifiers may be used tolabel the target molecules and/or target nucleic acids, for exampleorigin-specific barcodes and the like. The nucleic acid identifiers,nucleic acid barcodes, can include a short sequence of nucleotides thatcan be used as an identifier for an associated molecule, location, orcondition. In certain embodiments, the nucleic acid identifier furtherincludes one or more unique molecular identifiers and/or barcodereceiving adapters. A nucleic acid identifier can have a length ofabout, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, or 100 base pairs (bp) or nucleotides (nt). In certainembodiments, a nucleic acid identifier can be constructed incombinatorial fashion by combining randomly selected indices (forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes). Each suchindex is a short sequence of nucleotides (for example, DNA, RNA, or acombination thereof) having a distinct sequence. An index can have alength of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bp or nt. Nucleic acididentifiers can be generated, for example, by split-pool synthesismethods, such as those described, for example, in International PatentPublication Nos. WO 2014/047556 and WO 2014/143158, each of which isincorporated by reference herein in its entirety.

One or more nucleic acid identifiers (for example a nucleic acidbarcode) can be attached, or “tagged,” to a target molecule. Thisattachment can be direct (for example, covalent or noncovalent bindingof the nucleic acid identifier to the target molecule) or indirect (forexample, via an additional molecule). Such indirect attachments may, forexample, include a barcode bound to a specific-binding agent thatrecognizes a target molecule. In certain embodiments, a barcode isattached to protein G and the target molecule is an antibody or antibodyfragment. Attachment of a barcode to target molecules (for example,proteins and other biomolecules) can be performed using standard methodswell known in the art. For example, barcodes can be linked via cysteineresidues (for example, C-terminal cysteine residues). In other examples,barcodes can be chemically introduced into polypeptides (for example,antibodies) via a variety of functional groups on the polypeptide usingappropriate group-specific reagents (see for examplewww.drmr.com/abcon). In certain embodiments, barcode tagging can occurvia a barcode receiving adapter associate with (for example, attachedto) a target molecule, as described herein.

Target molecules can be optionally labeled with multiple barcodes incombinatorial fashion (for example, using multiple barcodes bound to oneor more specific binding agents that specifically recognizing the targetmolecule), thus greatly expanding the number of unique identifierspossible within a particular barcode pool. In certain embodiments,barcodes are added to a growing barcode concatemer attached to a targetmolecule, for example, one at a time. In other embodiments, multiplebarcodes are assembled prior to attachment to a target molecule.Compositions and methods for concatemerization of multiple barcodes aredescribed, for example, in International Patent Publication No. WO2014/047561, which is incorporated herein by reference in its entirety.

In some embodiments, a nucleic acid identifier (for example, a nucleicacid barcode) may be attached to sequences that allow for amplificationand sequencing (for example, SBS3 and P5 elements for Illuminasequencing). In certain embodiments, a nucleic acid barcode can furtherinclude a hybridization site for a primer (for example, asingle-stranded DNA primer) attached to the end of the barcode. Forexample, an origin-specific barcode may be a nucleic acid including abarcode and a hybridization site for a specific primer. In particularembodiments, a set of origin-specific barcodes includes a unique primerspecific barcode made, for example, using a randomized oligo typeNNNNNNNNNNNN.

A nucleic acid identifier can further include a unique molecularidentifier and/or additional barcodes specific to, for example, a commonsupport to which one or more of the nucleic acid identifiers areattached. Thus, a pool of target molecules can be added, for example, toa discrete volume containing multiple solid or semisolid supports (forexample, beads) representing distinct treatment conditions (and/or, forexample, one or more additional solid or semisolid support can be addedto the discreet volume sequentially after introduction of the targetmolecule pool), such that the precise combination of conditions to whicha given target molecule was exposed can be subsequently determined bysequencing the unique molecular identifiers associated with it.

Labeled target molecules and/or target nucleic acids associatedorigin-specific nucleic acid barcodes (optionally in combination withother nucleic acid barcodes as described herein) can be amplified bymethods known in the art, such as polymerase chain reaction (PCR). Forexample, the nucleic acid barcode can contain universal primerrecognition sequences that can be bound by a PCR primer for PCRamplification and subsequent high-throughput sequencing. In certainembodiments, the nucleic acid barcode includes or is linked tosequencing adapters (for example, universal primer recognitionsequences) such that the barcode and sequencing adapter elements areboth coupled to the target molecule. In particular examples, thesequence of the origin specific barcode is amplified, for example usingPCR. In some embodiments, an origin-specific barcode further comprises asequencing adaptor. In some embodiments, an origin-specific barcodefurther comprises universal priming sites. A nucleic acid barcode (or aconcatemer thereof), a target nucleic acid molecule (for example, a DNAor RNA molecule), a nucleic acid encoding a target peptide orpolypeptide, and/or a nucleic acid encoding a specific binding agent maybe optionally sequenced by any method known in the art, for example,methods of high-throughput sequencing, also known as next generationsequencing or deep sequencing. A nucleic acid target molecule labeledwith a barcode (for example, an origin-specific barcode) can besequenced with the barcode to produce a single read and/or contigcontaining the sequence, or portions thereof, of both the targetmolecule and the barcode. Exemplary next generation sequencingtechnologies include, for example, Illumina sequencing, Ion Torrentsequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencingamongst others. In some embodiments, the sequence of labeled targetmolecules is determined by non-sequencing based methods. For example,variable length probes or primers can be used to distinguish barcodes(for example, origin-specific barcodes) labeling distinct targetmolecules by, for example, the length of the barcodes, the length oftarget nucleic acids, or the length of nucleic acids encoding targetpolypeptides. In other instances, barcodes can include sequencesidentifying, for example, the type of molecule for a particular targetmolecule (for example, polypeptide, nucleic acid, small molecule, orlipid). For example, in a pool of labeled target molecules containingmultiple types of target molecules, polypeptide target molecules canreceive one identifying sequence, while target nucleic acid moleculescan receive a different identifying sequence. Such identifying sequencescan be used to selectively amplify barcodes labeling particular types oftarget molecules, for example, by using PCR primers specific toidentifying sequences specific to particular types of target molecules.For example, barcodes labeling polypeptide target molecules can beselectively amplified from a pool, thereby retrieving only the barcodesfrom the polypeptide subset of the target molecule pool.

A nucleic acid barcode can be sequenced, for example, after cleavage, todetermine the presence, quantity, or other feature of the targetmolecule. In certain embodiments, a nucleic acid barcode can be furtherattached to a further nucleic acid barcode. For example, a nucleic acidbarcode can be cleaved from a specific-binding agent after thespecific-binding agent binds to a target molecule or a tag (for example,an encoded polypeptide identifier element cleaved from a targetmolecule), and then the nucleic acid barcode can be ligated to anorigin-specific barcode. The resultant nucleic acid barcode concatemercan be pooled with other such concatemers and sequenced. The sequencingreads can be used to identify which target molecules were originallypresent in which discrete volumes.

Libraries and Cell Lines

The compositions of the present invention further include librariescomprising a multiplicity of the retroviral systems disclosed herein. Anumber of libraries may be used in accordance with the presentinvention, including but not limited to, normalized and non-normalizedlibraries for sense and antisense expression; libraries selected forspecific chromosomes or regions of chromosomes (e.g. as comprised inYACs or BACs), which would be possible by inclusion of the f1 origin;and libraries derived from a tissue source; and genomic libraries.

In some cases, the compositions herein comprise a viral expressionlibrary. The viral expression library may comprise viral particles,wherein each viral particle comprises a polynucleotide having engineeredassociations comprising a sequence encoding one or more geneticperturbations and a unique molecular sequence clone, and one or morepolypeptides that comprise non-recombinogenic RNA sequences, or proteinsthat are capable of dimerizing with the polynucleotide.

The libraries employed in embodiments of the subject methods can beproduced using any convenient protocol. According to certainembodiments, preparing the libraries includes combining polynucleotidehaving a first engineered association and a second engineeredassociation with a vector construct comprising a vector domain of vectorsequence under conditions sufficient to produce transfection plasmidswhich, upon transfection of a packaging cell, result in the productionof viral particles containing the polynucleotide as part of genomicnucleic acids encapsidated in viral protein shells. To prepare theproduct transfection plasmids used for transfection, a polynucleotidemay be inserted into a vector nucleic acid, where any suitable protocolmay be employed. Examples of suitable protocols include, but are notlimited to: DNA ligase mediated joining, recombination enzyme mediatejoining, Gateway® cloning technology (Life Technologies, Carlsbad,Calif.), and the like.

The resultant product transfection plasmids may then be used totransfect a suitable packaging cell line for production of library viralparticles. The packaging cell line provides the viral proteins that arerequired in trans for the packaging of the viral genomic RNA into viralparticles. The packaging cell line may be any cell line that is capableof expressing retroviral proteins, including HEK293, HeLa, D17, MDCK,BHK, NIH3T3, CHO, CrFK, and Cf2Th. In some embodiments, the construct isused together with a viral reporter construct which may comprise one ormore reporter genes under the control of a constitutive or conditionalpromoter. The packaging cell line may stably express necessary viralproteins. Such a packaging cell line is described, for example, in U.S.Pat. No. 6,218,181. Alternatively, a packaging cell line may betransiently transfected with plasmids comprising nucleic acids thatencode the necessary viral proteins. In another embodiment, a packagingcell line that does not stably express the necessary viral proteins isco-transfected with two or more plasmids. One of the plasmids comprisesthe viral construct comprising the polynucleotide. The other plasmid(s)comprises nucleic acids encoding the proteins necessary to allow thecells to produce functional virus that is able to infect the desiredhost cell. The packaging cell line may not express envelope geneproducts. In this case, the packaging cell line will package the viralgenome into particles that lack an envelope protein. As the envelopeprotein is responsible, in part, for the host range of the viralparticles, the viruses preferably are pseudotyped. A “pseudotyped”retrovirus is a retroviral particle having an envelope protein that isfrom a virus other than the virus from which the RNA genome is derived.The envelope protein may be from a different retrovirus or anon-retrovirus. One envelope protein is the vesicular stomatitis virus G(VSV-G) protein. Thus, the packaging cell line may be transfected with aplasmid that includes sequences encoding a membrane-associated protein,such as VSV-G, that will permit entry of the virus into a target cell.One of skill in the art can choose an appropriate pseudo type specificand/or more efficient for the target cell used. In addition toconferring a specific host range, a chosen pseudotype may permit thevirus to be concentrated to a very high titer. Viruses alternatively canbe pseudotyped with ecotropic envelope proteins that limit infection toa specific species.

The compositions of the present invention further include retrovirusparticles derived from said first and second polynucleotides and otherpackaging vectors needed to form a complete viral particle. Suchretrovirus particles are produced by the transfection of thepolynucleotides and/or packaging vectors into retroviral cell packagingcell lines. Thus stably transfected cell lines comprising said sequencesare also within the scope of the invention disclosed herein. Thecompositions of the invention further include provirus sequences derivedfrom the retrovirus particles. The provirus sequences may be present inan integrated form within the genome of a recipient cell, or may bepresent in a free, circularized form. An integrated provirus is producedupon infection of a recipient cell, wherein the infection leads to theproduction an integration into the cell genome of the provirus nucleicacid sequence. The circularized provirus sequence may generally beproduced upon excision of the integrated provirus from the recipientcell genome.

The compositions of the present invention still further include cellscontaining the retroviral systems disclosed herein, whether thepackaging cell lines or recipient cell lines. Additionally, the presentinvention includes transgenic animals containing the retroviral systemsdisclosed herein, including preferably animals containing retroviralsystems form which sequences (sense or antisense) are expressed in oneor more cells.

Methods for Making Lentiviral System

In one aspect, the embodiments disclosed herein are directed to methodsof preparing a lentiviral or retroviral system comprising apolynucleotide having engineered associations comprising a sequenceencoding one or more genetic perturbations and a unique molecularsequence wherein the system has reduced recombination or templateswitching, activity. The methods may comprise packaging thepolynucleotide with a modulator of one or more activities of the system.The modulator may be an inhibitor of recombination or template switchingactivity. For example, the modular may be an inhibitor of templateswitching. In one embodiment, polynucleotides encoding the one or moregenetic perturbation and associated unique molecular sequence are clonedinto a suitable lentiviral or retroviral vector (“targeting vector”).Suitable vectors include, for example; pBA571 (Addgene Cat #85968),pMJ114 (Addgene Cat #85995), pMJ179 (Addgene Cat #85996), pMJ1117(Addgene Cat #85997). Carrier plasmids are likewise selected. Thecarrier plasmids do not include sequences encoding the one or moregenetic perturbations or the unique molecular sequence. Instead carrierplasmids are selected to comprise non-recombinogenic sequences, orencode proteins that are capable of dimerizing with the polynucleotideof sequence. Example carrier polynucleotides include pr_H2b-BFB(encoding a hi stone subunit tagged with blue fluorescent protein) andpLX_TRC131_LacZ (control vector used in ORF screens). In certainembodiments, the carrier plasmid may comprise a lentiviral or retroviralplasmid that has been modified to be non-integrating. For example, alentiviral vector may be made non-integrating by mutating the 5′ longterminal repeat (LTR) and having a short LTR to LTR distance of 2.1 kb.Example proteins that are capable of dimerizing are disclosed inretroviral nucleoproteins (NC). The target vector and carrier may thenbe introduced along with standard lentiviral or retroviral packagingplasmids that encode remaining elements need for full viral particleproduction into a packaging cell lines to generate a viral clonelibrary, each clone comprising a different target vector and one or morecarrier vectors. The target vector may be diluted in a composition withone or more carrier vectors prior to introduction in the packaging cellline. In certain example embodiments, the target vector is diluted in asolution comprising one or more carrier vectors prior to introductioninto the packaging cell line at a dilution of 1:10, 1:20, 1:30, 1:40,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600,1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, or 1:5000.

Also provided herein include a cell or cell line for the viral systemsherein. The cell or cell line may be used for producing viral particles.In some cases, the compositions may comprise a cell or cell line forproducing viral particles comprising a set of polynucleotide constructssuch that the viral particles comprise polynucleotides having engineeredassociations comprising a sequence encoding one or more geneticperturbations and a unique molecular sequence clone, and one or morepolypeptides that comprise non-recombinogenic RNA sequences, or proteinsthat are capable of dimerizing with the polynucleotide.

Genetic Screens

In some embodiments, the present disclosure includes methods forscreening cells for genetic perturbations. The methods may comprise oneor more of: (i) providing (e.g., culturing) a cell or population ofcells in one or more discrete volumes; introducing the system describedherein, such that each cell receives one or more polynucleotides eachhaving at least one genetic perturbation and a unique identifier;detecting genomic, genetic, proteomic, epigenetic and/or phenotypicdifferences in single cells; and identifying the at least one geneticperturbation in each cell based on the unique identifier.

In one aspect, the present invention provides for a method ofreconstructing a cellular network or circuit, comprising introducing atleast 1, 2, 3, 4 or more single-order or combinatorial perturbations toa plurality of cells in a population of cells, wherein each cell in theplurality of the cells receives at least 1 perturbation; measuringcomprising: detecting genomic, genetic, proteomic, epigenetic and/orphenotypic differences in single cells compared to one or more cellsthat did not receive any perturbation, and detecting the perturbation(s)in single cells; and determining measured differences relevant to theperturbations by applying a model accounting for co-variates to themeasured differences, whereby intercellular and/or intracellularnetworks or circuits are inferred. The measuring in single cells maycomprise single cell sequencing. The single cell sequencing may comprisecell barcodes, whereby the cell-of-origin of each RNA is recorded. Thesingle cell sequencing may comprise unique molecular identifiers (UMI),whereby the capture rate of the measured signals, such as transcriptcopy number or probe binding events, in a single cell is determined. Themodel may comprise accounting for the capture rate of measured signals,whether the perturbation actually perturbed the cell (phenotypicimpact), the presence of subpopulations of either different cells orcell states, and/or analysis of matched cells without any perturbation.

The single-order or combinatorial perturbations may comprise 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100 perturbations. The perturbation(s) may target genes in apathway or intracellular network.

The measuring may comprise detecting the transcriptome of each of thesingle cells. The perturbation(s) may comprise one or more geneticperturbation(s). The perturbation(s) may comprise one or more epigeneticor epigenomic perturbation(s). At least one perturbation may beintroduced with RNAi- or a CRISPR-Cas system. At least one perturbationmay be introduced via a chemical agent, biological agent, anintracellular spatial relationship between two or more cells, anincrease or decrease of temperature, addition or subtraction of energy,electromagnetic energy, or ultrasound.

The cell(s) may comprise a cell in a model non-human organism, a modelnon-human mammal that expresses a Cas protein, a mouse that expresses aCas protein, a mouse that expresses Cpf1, a cell in vivo or a cell exvivo or a cell in vitro. The cell(s) may also comprise a human cell.

The measuring or measured differences may comprise measuring or measureddifferences of DNA, RNA, protein or post translational modification; ormeasuring or measured differences of protein or post translationalmodification correlated to RNA and/or DNA level(s).

The perturbing or perturbation(s) may comprise(s) genetic perturbing.The perturbing or perturbation(s) may comprise(s) single-orderperturbations. The perturbing or perturbation(s) may comprise(s)combinatorial perturbations. The perturbing or perturbation(s) maycomprise gene knock-down, gene knock-out, gene activation, geneinsertion, or regulatory element deletion. The perturbation may resultin a change. The perturbing or perturbation(s) may comprise genome-wideperturbation. The perturbing or perturbation(s) may comprise performingCRISPR-Cas-based perturbation. The perturbing or perturbation(s) maycomprise performing pooled single or combinatorial CRISPR-Cas-basedperturbation with a genome-wide library of sgRNAs. The perturbations maybe of a selected group of targets based on similar pathways or networkof targets.

The perturbing or perturbation(s) may comprises performing pooledcombinatorial CRISPR-Cas-based perturbation with a genome-wide libraryof sgRNAs. Each sgRNA may be associated with a unique perturbationbarcode. Each sgRNA may be co-delivered with a reporter mRNA comprisingthe unique perturbation barcode (or sgRNA perturbation barcode).

The perturbing or perturbation(s) may comprise subjecting the cell to anincrease or decrease in temperature. The perturbing or perturbation(s)may comprise subjecting the cell to a chemical agent. The perturbing orperturbation(s) may comprise subjecting the cell to a biological agent.The biological agent may be a toll like receptor agonist or cytokine.The perturbing or perturbation(s) may comprise subjecting the cell to achemical agent, biological agent and/or temperature increase or decreaseacross a gradient.

The cell may be in a microfluidic system. The cell may be in a droplet.The population of cells may be sequenced by using microfluidics topartition each individual cell into a droplet containing a uniquebarcode, thus allowing a cell barcode to be introduced.

The perturbing or perturbation(s) may comprise transforming ortransducing the cell or a population that includes and from which thecell is isolated with one or more genomic sequence-perturbationconstructs that perturbs a genomic sequence in the cell. Thesequence-perturbation construct may be a viral vector, preferably alentivirus vector. The perturbing or perturbation(s) may comprisemultiplex transformation or transduction with a plurality of genomicsequence-perturbation constructs.

In another aspect, or in alternative embodiments of aspects describedherein, the present invention provides for a method wherein proteins ortranscripts expressed in single cells are determined in response to aperturbation, wherein the proteins or transcripts are detected in thesingle cells by binding of more than one labeling ligand comprising anoligonucleotide tag, and wherein the oligonucleotide tag comprises aunique constituent identifier (UCI) specific for a target protein ortranscript. The single cells may be fixed in discrete particles. Thediscrete particles may be washed and sorted, such that cell barcodes maybe added, e.g. sgRNA perturbation barcodes as described above. Theoligonucleotide tag and sgRNA perturbation barcode may comprise auniversal ligation handle sequence, whereby a unique cell barcode may begenerated by split-pool ligation. The labeling ligand may comprise anoligonucleotide label comprising a regulatory sequence configured foramplification by T7 polymerase. The labeling ligands may compriseoligonucleotide sequences configured to hybridize to a transcriptspecific region. Not being bound by a theory, both proteins and RNAs maybe detected after perturbation. The oligonucleotide label may furthercomprise a photocleavable linker. The oligonucleotide label may furthercomprise a restriction enzyme site between the labeling ligand andunique constituent identifier (UCI). The ligation handle may comprise arestriction site for producing an overhang complementary with a firstindex sequence overhang, and wherein the method further comprisesdigestion with a restriction enzyme. The ligation handle may comprise anucleotide sequence complementary with a ligation primer sequence andwherein the overhang complementary with a first index sequence overhangis produced by hybridization of the ligation primer to the ligationhandle. The method may further comprise quantitating the relative amountof UCI sequence associated with a first cell to the amount of the sameUCI sequence associated with a second cell, whereby the relativedifferences of a cellular constituent between cell(s) are determined.The labeling ligand may comprise an antibody or an antibody fragment.The antibody fragment may be a nanobody, Fab, Fab′, (Fab′)2, Fv, ScFv,diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3fragment. The labeling ligand may comprise an aptamer. The labelingligand may be a nucleotide sequence complementary to a target sequence.

Single cell sequencing may comprise whole transcriptome amplification.

The method in aspects of the invention may comprise comparing an RNAprofile of the perturbed cell with any mutations in the cell to alsocorrelate phenotypic or transcriptome profile and genotypic profile.

In another aspect, or in alternative embodiments of aspects describedherein, the present invention provides for a method comprisingdetermining genetic interactions by causing a set of P geneticperturbations in single cells of the population of cells, wherein themethod comprises: determining, based upon random sampling, a subset of πgenetic perturbations from the set of P genetic perturbations;performing said subset of π genetic perturbations in a population ofcells; performing single-cell molecular profiling of the population ofgenetically perturbed cells; inferring, from the results and based uponthe random sampling, single-cell molecular profiles for the set of Pgenetic perturbations in cells. The method may further comprise: fromthe results, determining genetic interactions. The method may furthercomprise: confirming genetic interactions determined with additionalgenetic manipulations.

The set of P genetic perturbations or said subset of π geneticperturbations may comprise single-order genetic perturbations. The setof P genetic perturbations or said subset of π genetic perturbations maycomprise combinatorial genetic perturbations. The genetic perturbationmay comprise gene knock-down, gene knock-out, gene activation, geneinsertion, or regulatory element deletion. The set of P geneticperturbations or said subset of π genetic perturbations may comprisegenome-wide perturbations. The set of P genetic perturbations or saidsubset of π genetic perturbations may comprise k-order combinations ofsingle genetic perturbations, wherein k is an integer ranging from 2 to15, and wherein the method comprises determining k-order geneticinteractions. The set of P genetic perturbations may comprisecombinatorial genetic perturbations, such as k-order combinations ofsingle-order genetic perturbations, wherein k is an integer ranging from2 to 15, and wherein the method comprises determining j-order geneticinteractions, with j<k.

The method in aspects of this invention may comprise performing RNAi- orCRISPR-Cas-based perturbation. The method may comprise an array-formator pool-format perturbation. The method may comprise pooled single orcombinatorial CRISPR-Cas-based perturbation with a genome-wide libraryof sgRNAs. The method may comprise pooled combinatorial CRISPR-Cas-basedperturbation with a genome-wide library of sgRNAs.

The random sampling may comprise matrix completion, tensor completion,compressed sensing, or kernel learning. The random sampling may comprisematrix completion, tensor completion, or compressed sensing, and whereinπ is of the order of log P.

The cell may comprise a eukaryotic cell. The eukaryotic cell maycomprise a mammalian cell. The mammalian cell may comprise a human cell.The cell may be from a population comprising 10² to 10⁸ cells and DNA orRNA or protein or post translational modification measurements orvariables per cell comprise 50 or more.

The perturbation of the population of cells may be performed in vivo.The perturbation of the population of cells may be performed ex vivo andthe population of cells may be adoptively transferred to a subject. Thepopulation of cells may comprise tumor cells. The method may comprise alineage barcode associated with single cells, whereby the lineage orclonality of single cells may be determined.

The perturbing may be across a library of cells to thereby obtain RNAlevel and/or optionally protein level, whereby cell-to-cell circuit dataat genomic or transcript or expression level is determined. The libraryof cells may comprise or is from a tissue sample. The tissue sample maycomprise or is from a biopsy from a mammalian subject. The mammaliansubject may comprise a human subject. The biopsy may be from a tumor.The method may further comprise reconstructing cell-to-cell circuits.

The method may comprise measuring open chromatin and may comprisefragmenting chromatin inside isolated intact nuclei from a cell, addinguniversal primers at cutting sites, and uniquely tagging DNA thatoriginated from the cell.

The method may comprise measuring protein and RNA levels and maycomprise CyTOF.

In another aspect, the present invention provides for a method ofdetermining any combination of protein detection, RNA detection, openchromatin detection, protein-protein interactions, protein-RNAinteractions, or protein-DNA interactions comprising any of thepreceding methods.

In another aspect, the present invention provides for a method forscreening compounds or agents capable of modifying a cellular network orcircuit comprising performing any method as described herein, whereinperturbing further comprises exposing the cell to each compound oragent.

In another aspect, the present invention provides for a method ofidentifying a therapeutic, and to a therapeutic identified by the methoddescribed herein.

In another aspect, the present invention provides a method ofreconstructing a cellular network or circuit, comprising introducing atleast 1, 2, 3, 4 or more single-order or combinatorial perturbations toeach cell in a population of cells; measuring genomic, genetic and/orphenotypic differences of each cell and coupling combinatorialperturbations with measured differences to infer intercellular and/orintracellular networks or circuits. The single-order or combinatorialperturbations can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 or massively parallelperturbations. The perturbation(s) can comprise one or more geneticperturbation. The perturbation(s) can comprise one or more epigenetic orepigenomic perturbation. The perturbation can be introduced with RNAi-or a CRISPR-Cas system. For example, reference is also made to Dahlmanet al., Nature Biotechnology (2015) doi:10.1038/nbt.3390 Publishedonline 5 Oct. 2015 to allow efficient orthogonal genetic and epigeneticmanipulation. Dahlman et al., Nature Biotechnology (2015)doi:10.1038/nbt.3390 have developed a CRISPR-based method that usescatalytically active Cas9 and distinct single guide (sgRNA) constructsto knock out and activate different genes in the same cell. ThesesgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, canactivate gene expression using an active Streptococcus pyogenes Cas9nuclease, without inducing double-stranded breaks. Dahlman et al.,Nature Biotechnology (2015) doi:10.1038/nbt.3390 use these ‘dead RNAs’to perform orthogonal gene knockout and transcriptional activation inhuman cells.

The at least one perturbation can be introduced via a chemical agent, anintracellular spatial relationship between two or more cells, anincrease or decrease of temperature, addition or subtraction of energy,electromagnetic energy, or ultrasound. The cell can comprise a cell in amodel non-human organism, a model non-human mammal that expresses a Casprotein, a mouse that expresses a Cas protein, a cell in vivo or a cellex vivo or a cell in vitro. The measuring or measured differences cancomprise measuring or measured differences of DNA, RNA, protein or posttranslational modification; or measuring or measured differences ofprotein or post translational modification correlated to RNA and/or DNAlevel(s). The method can include sequencing, and prior to sequencing:perturbing and isolating a single cell with at least one labeling ligandspecific for binding at one or more target RNA transcripts, or isolatinga single cell with at least one labeling ligand specific for binding atone or more target RNA transcripts and perturbing the cell; and/orlysing the cell under conditions wherein the labeling ligand binds tothe target RNA transcript(s).

The method in aspects of this invention may also include, prior tosequencing perturbing and isolating a single cell with at least onelabeling ligand specific for binding at one or more target RNAtranscripts, or isolating a single cell with at least one labelingligand specific for binding at one or more target RNA transcripts andperturbing the cell; and lysing the cell under conditions wherein thelabeling ligand binds to the target RNA transcript(s). The perturbingand isolating a single cell may be with at least one labeling ligandspecific for binding at one or more target RNA transcripts. Theisolating a single cell may be with at least one labeling ligandspecific for binding at one or more target RNA transcripts andperturbing the cell.

The perturbing of the present invention may involve genetic perturbing,single-order genetic perturbations or combinatorial geneticperturbations. The perturbing may also involve gene knock-down, geneknock-out, gene activation, gene insertion or regulatory elementdeletion. The perturbation may be genome-wide perturbation. Theperturbation may be performed by RNAi- or CRISPR-Cas-based perturbation,performed by pooled single or combinatorial CRISPR-Cas-basedperturbation with a genome-wide library of sgRNAs or performing pooledcombinatorial CRISPR-Cas-based perturbation with a genome-wide libraryof sgRNAs.

In addition to loss-of-function (LOF) mutations, embodiments disclosedherein may also be used to modulate transcription without modifyinggenomic sequences. For example, inactive Cas9 (dCas9) can becatalytically fused to transcriptional activation and repressiondomains. CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) canbe achieved by direct fusion or recruitment of activation and repressiondomains, such as VP64 and KRAB, respectively. Methods for setting up GOFand LOF genetic screens are described in detail in Joung et al. NatProtoc. 2017 April: 12(4): 828-863.

Methods and tools for genome-scale screening of perturbations in singlecells using CRISPR-Cas9 have been described, herein referred to asperturb-seq (see e.g., Dixit et al., “Perturb-Seq: Dissecting MolecularCircuits with Scalable Single-Cell RNA Profiling of Pooled GeneticScreens” 2016, Cell 167, 1853-1866; Adamson et al., “A MultiplexedSingle-Cell CRISPR Screening Platform Enables Systematic Dissection ofthe Unfolded Protein Response” 2016, Cell 167, 1867-1882; andInternational publication serial number WO/2017/075294). The presentinvention is compatible with perturb-seq, such that lentiviral vectorstargeting genes for perturbation may be identified and assigned to theproteomic and gene expression readouts of single cells based ontranscripts encoding for guide sequence specific barcodes. The presentinvention can be used to prevent recombination during packaginglentiviral libraries that may shuffle associations between guidesequences and barcode transcripts, thus greatly improving phenotypicreadouts associated with a perturbation.

Methods for Reducing Intermolecular Recombination

In some embodiments, the present disclosure provides methods forreducing intermolecular recombination with a lentiviral genome plasmidof interest in a library (e.g., lentiviral library). The methods maycomprise mixing the lentiviral genome plasmid of interest with alentiviral carrier plasmid, and packaging the mixture. The lentiviralcarrier plasmid may comprise a non-integrating lentiviral vector, anon-recombinogenic lentiviral vector, or a combination thereof. Thelibrary herein may comprise a barcode library, a plurality of guidepolynucleotides, a plurality of sgRNAs, or any combination thereof.

The lentiviral genome plasmid of interest and the lentiviral carrierplasmid may be mixed at a suitable ratio. The ratio of the lentiviralgenome plasmid of interest to the lentiviral carrier plasmid may be atleast 1:1, 2:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 60:1, 70:1,80:1, 90:1, 100:1, 120:1, 150:1, or 200:1.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1

Lentiviral vectors provide a convenient, scalable platform to delivergenetic perturbations to cells en masse and read out the identity ofeach perturbation by next-generation sequencing^(1, 2). There isincreasing interest in screening approaches reliant on the delivery ofmultiple library elements to each cell, for example, in CRISPR-basedsingle-cell gene expression screens³⁻⁸. Such approaches facilitate thestudy of genetic interactions by probing cells with combinations ofperturbations or convenient detection of perturbations by readout of abarcode sequence. However, the goal of accurately delivering a singleintegrated library variant per cell is complicated by aspects oflentiviral delivery.

Lentiviral virions normally contain two copies of the viral genome.During standard lentivirus production, transfection of packaging cellswith multiple plasmids generates virions containing two distinct libraryelements, which can then lead to intermolecular recombination thatshuffles variable library sequences (“barcode swapping”). Together withthe inadvertent integration of multiple variants in individual targetcells, this process has the effect of reducing the sensitivity of pooledscreens. For screens where all library elements are read out (e.g.targeted pairs of gene knockouts), recombination events can be detectedand filtered out before statistical analysis^(6, 9). However, insituations where functional library elements are not sequenced directly,but are rather inferred via a linked barcode, recombination or multipleintegration can lead to mislabeled data and has been noted to decreasethe statistical power of genetic screens at a given number of cellsanalyzed^(10, 11).

Recombination can arise from the template-switching activity of thelentiviral reverse-transcriptase¹². As the lentivirus capsid normallypackages a dimer of RNA genomes, intermolecular recombination could inprinciple occur in target cells infected by a single virion. Thefraction of target cells with recombined integrants depends on thedistance between variable sequences and has been measured to exceed 30%for distances greater than 1 kb¹³. Such wide spacing of library elementsis common when the elements are separated by regulatory sequences orwhen an element is used as a 3′ barcode in an expressedtranscript^(10, 11, 13).

To quantify the frequencies of barcode swapping and multiple integrationevents, we performed clonal analysis of target cells transduced with alibrary of CRISPR sgRNA and transcribed barcode elements separatedby >1.7 kb. Similar to other groups, Applicant found that standardlentiviral packaging results in substantial (>30%) barcode swappingbetween library elements. Applicant further observed that anunexpectedly high number of target cells had multiple library variantsintegrated into their genomes even when transduced at lowmultiplicity-of-infection (MOI). Here Applicant shows that by dilutingthe perturbation library plasmid with sufficient excess of carrierplasmid in the packaging step, Applicant was able to substantiallyreduce barcode swaps (<4%) and attenuate the rate of multipleintegrations several-fold. Altogether, this co-packaging strategyconstitutes a simple solution to improve data quality for geneticscreens without constraining library vector design or necessitatingindividual (“arrayed”) packaging of library element.

Results

In order to test the feasibility of barcoding a U6-driven sgRNA with ashort sequence located in the 3′ UTR of a Pol II-driven resistancetranscript, Applicant individually cloned 8 lentiviral plasmids withdifferent sgRNA-barcode pairs and transduced HeLa cells at an MOI<5%,pooling either before or after lentiviral packaging. After flow sortingand clonally expanding single cells, Applicant analyzed the sgRNA andbarcode sequences present in each clone using next-generation sequencing(Methods). Applicant observed that pooled lentiviral packaging resultedin barcode swaps in 37% of clones with a single detected integration,whereas no swapping was detected between individually packagedlentiviral genome sequences (Table 1). Similar results were obtainedwhen packaging a library of 400 barcodes (cloned as a pool) using thestandard protocol. Applicant reports barcode swapping rates as thefunctional and measurable outcome of recombination. Overall, the resultsare consistent with observations by a number of groups and precautionarycomments published in some of the first examples of pooled single-cellgene expression screens^(10, 11, 14).

The standard pooled lentivirus packaging protocol calls for transfectingthe packaging cell line with the lentiviral perturbation library andassociated packaging plasmids needed to produce virus (Methods). Thepresent clonal analysis of target cells transduced by virus produced inthis manner revealed not only barcode swaps but also a higher occurrenceof multiple integrants per cell than predicted by a Poisson model ofindependent integration events, even at MOI below 5%. The presence ofmultiple genomic integrants even at limiting virus dilutions could beexplained by the ability of a single virion to integrate both packagedgenomes, or non-independence of integration probability across targetcells, possibly as a result of differences in cell state.

Hypothesizing that recombination and multiple integration events aredriven by co-packaging of two RNA genomes per lentiviral capsid¹³ andco-delivery of multiple genomes to individual target cells, Applicanttested dilution of the perturbation library plasmids in unrelatedcarrier plasmids as a means of mitigating both of these undesiredeffects. Three lentiviral plasmids were evaluated as carriers, includingtwo integration-capable vectors (pR_H2B-BFP encoding a histone subunittagged with blue fluorescent protein, and pLX_TRC313_LacZ, a controlvector used in ORF screens). In addition, Applicant tested anon-integrating lentiviral vector with a mutated 5′ long terminal repeat(LTR) and a short LTR-LTR distance of 2.1 kb in hopes of avoidingunnecessary genomic integrations¹⁵. All three carriers tested reducedrecombination rates to 0-4%. Interestingly, Applicant found that whileit was necessary to use the integrating carrier plasmids in 1.000-foldexcess over the perturbation library, the non-integrating carrierplasmid reduced barcode swaps to the same extent at a dilution of only1:10, perhaps due to enhanced expression of the shorter LTR-LTRtranscript in the packaging cell line. Furthermore, co-packaging with anon-homologous lentiviral vector also limited instances of multipledistinct integrations, likely due to a reduction in the probability thattwo perturbation library variants enter the target cell.

Applicant also tested dilution in a non-lentiviral carrier plasmid,pUC19, hypothesizing that stringent dilution of the perturbation librarycould reduce the number of library, variants in each packaging cell toone or fewer and minimize the risk that heterodimeric virions areproduced. This strategy was found to decrease the recombination rate to6%. However, Applicant still observed 18% of cell clones with greaterthan one integrated library variant, consistent with the correlatedinfection hypothesis and indicating that lentiviral plasmids may be abetter choice of carrier material.

A limitation of this dilution strategy is a 100-fold decrease in titerrelative to lentivirus prepared with the non-diluted perturbationlibrary, measured by counting the number of cell colonies survivingafter antibiotic selection. To investigate the trade-off between titerand unwanted lentiviral effects, Applicant titrated the dilution ratioof one of the integrating lentiviral carrier plasmids (pLX_TRC313_LacZ)but found that 100-fold excess did not show the desired performance,with 25% of colonies showing barcode swaps or multiple integrants.Nevertheless, even with diminished viral titer, Applicant was able totransduce a library of 1,000 perturbations with 300-fold cell coverage.

Applicant also explored whether recombination events hypotheticallyoccurring in the packaging cell line could be reduced by shorteningpackaging times. The time from transfection to harvesting viralsupernatant was reduced from 48 h to 11 h, a decrease in barcodeswapping was not observed, consistent with a model where most of therecombination occurs in the target cells.

TABLE 1 Clonal analysis of lentiviral packaging strategies for barcodedperturbation libraries. Individual transduced cells were isolated byflow sorting and clonally expanded prior to genomic DNA extraction andexamination of sgRNA and barcode identity by next-generation sequencing.Each clone was classified by the number of observed integrations, withsingle integrants further subdivided by whether the sgRNA matched theassociated barcode. A recombination rate (rightmost column) wasestimated by dividing the number of recombined single integrants by thetotal number of cells with a single integration. Standard packagingrefers to transfection of the pooled perturbation library alone withpackaging plasmids (pMD2.G and psPAX2); for arrayed packaging, eachlibrary element was individually co-transfected with packaging plasmidsfor production of a pure population of virions that was subsequentlypooled. Carrier plasmids come in three varieties: non-lentiviral(pUC19), integrating lentiviral (pR_H2B- BFP and pLX_TRC313_LacZ) andnon-integrating lentiviral (pR_LG). Finally, a quick harvest (11 hours)of the packaged lentivirus was also explored; for all other conditions,virus was packaged for 48 hours # of cell Single Estimated # of clonesSingle integration, integration, Multiple barcode Relative Packagingcondition barcodes analyzed correct association barcode swap integrationswap rate titer arrayed 8 48 46 (95.8%) 0 (0.0%) 2 (4.2%)    0% 100%standard 8 61 34 (55.7%) 20 (32.8%) 7 (11.5%) 37.0%  100% standard 40028 16 (57.1%)  8 (28.6%) 4 (14.3%) 33.3%  100% 10x dilution in pR_LG 40068 67 (98.5%) 1 (1.5%) 0 (0.0%)  1.5% 1% 1000x dilution in pR_H2B-BFP 861 59 (96.7%) 0 (0.0%) 2 (3.3%)    0% 1% 1000x dilution inpLX_TRC313_LacZ 400 53 50 (94.3%) 2 (3.8%) 1 (1.9%)  3.8% 1% 100xdilution in pLX_TRC313_Lacz 400 16 12 (75.0%) 1 (6.2%) 3 (18.8%) 7.7% 3%1000x dilution in pUC19 8 45 35 (77.8%) 2 (4.4%) 8 (17.8%) 5.4% 1%standard, quick harvest (11 h) 8 51 28 (54.9%) 17 (33.3%) 6 (11.8%)37.8%  3%

In the context of genetic screens, lentiviral co-packaging can greatlyreduce barcode swaps and decrease the background of multiple integrantswithout constraining library vector design or necessitating individualpackaging of library elements. The latter approach was used by Adamsonet al. to avoid recombination in a single-cell gene expression screen,but is limited in scalability¹⁴. Datlinger et al. developeda—specialized CROP-seq vector in which the sgRNA is itself transcribedby Pol II and captured in a 3′ RNA-seq protocol, obviating the need foran additional barcode and eliminating concerns about recombination⁵.However, this approach requires locating the perturbation within the 3′LTR and is not generalizable to some types of screens (e.g. pairedperturbation screens, or screens of regulatory elements monitored viatranscribed barcodes)¹⁶. By addressing both recombination and multipleintegration, co-packaged lentiviral libraries have the potential toimprove the accuracy of perturbation barcoding and boost the sensitivityof screens that deliver library constructs with multiple variableelements.

Applicant chose to employ clonal analysis of the genomic integrants bynext-generation sequencing to achieve sensitive and unbiased detectionof perturbation library elements with single-cell resolution. AsApplicant amplified each variable sequence in a separate PCR reaction,this approach is not subject to artifacts resulting from PCR-basedrecombination. Moreover, the readout does not depend on eventssubsequent to integration and antibiotic selection, such as detection ofa fluorescent marker or perturbation of cellular phenotype that may beconfounded by multiple integrations. However, clonal analysis ispractically limited to the scale of 100-1000 clones per sequencing run,making it better suited for high-confidence measurements of undesiredintegration events than for systematic optimization across many testconditions.

Under the standard assumption of a zero-truncated Poisson distributionof lentiviral integrations, one would expect a multiple integration ratebelow 2.5% when transducing cells at an MOI below 5%. However, despiteworking in this range for our infections, the measured multipleintegration rate was greater than 10%, suggesting that, at least in ourHeLa cell system, lentiviral integrations detected after antibioticselection are correlated and do not follow a zero-truncated Poissondistribution as is commonly assumed¹⁷. It is likely that multipleintegration background is a persistent noise source in genetic screensutilizing lentivirus, with an effect that depends on the particularsystem. A lack of statistical independence between integration eventsunderscores the need to maintain high representation of library elementsin transduced cells in order to average over technical and biologicalnoise.

Decreased viral titer is a potential drawback to the dilute co-packagingapproach. Here, Applicant opted to perform clonal analysis understringent dilution conditions, which minimized recombination butexacerbated the viral titer issue. For example, using theintegration-defective pR_LG 1:10 co-packaging condition, Applicant wasable to generate 10,000 HeLa colonies per mL of viral supernatant. Atthis titer, 35 mL of viral supernatant would be sufficient for onereplicate of a screen involving 1,000 perturbations with 300 initiallyinfected cells per library element. The cost of tissue culture andtransfection reagents to generate 35 mL of viral supernatant iscurrently orders of magnitude smaller than the cost of preparing andsequencing single-cell gene expression libraries covering 1,000perturbations and does not thus pose a limit to the achievable scale ofthe screen. On the other hand, this titer may be prohibitive for agenome-wide CRISPR screen reading out enrichment of more than 50,000sgRNAs by amplicon sequencing. Ultimately, any particular screen willexhibit an optimal trade-off between degradation of data quality bylentiviral recombination and loss of titer due to co-packaging with acarrier plasmid, and the user may select an appropriate level ofdilution to balance these effects.

The ability to minimize both recombination and multiple integrations bydiluting the transfer vector in another lentiviral plasmid supports thehypothesis that RNA dimerization during lentiviral packaging is involvedin these undesired outcomes. In the case of dilution with excesslentiviral carrier plasmid, most library genomes are likely packagedwith a non-homologous carrier genome such that the frequency of virionscontaining two library variants is substantially reduced. Meanwhile,limiting dilution with a non-lentiviral carrier plasmid may reduce thelikelihood that two library variants are transfected into the same cellfor packaging, hence preventing two different library RNA genomes fromdimerizing. Both of these approaches mitigate the potential for templateswitching and recombination between two different library genomes in thetarget cells.

Alternative approaches to control recombination include directlyinhibiting the template-switching activity of the viral reversetranscriptase in the transduced cells or biasing packaging to a singlegenome per virion by modifying the RNA sequence or proteins involved indimerization¹⁸. Such efforts could potentially address both the effectsof recombination and multiple genomic integrations without acorresponding loss in titer due to dilution.

Methods

Single cell clonal analysis of integrated sgRNAs and barcodes wasperformed by transducing a lentiviral library into a target cellpopulation, selecting with antibiotic, sorting and expanding singlecells, and separately PCR-amplifying and deep sequencing the sgRNA andbarcode sequences from each expanded colony. All cell lines weretransduced at an MOI<5%, determined by counting the fraction of cellssurviving antibiotic selection.

Lentivirus was prepared following published methods¹⁹. All cell cultureused Dulbecco's Modified Eagle's Medium supplemented with 10% FBS (GELife Sciences SH30070.03T), 100 units/mL penicillin, and 100 μg/mLstreptomycin. A 4:3:2 ratio by mass of packaging plasmids pMD2.G(Addgene 12259) and psPAX2 (Addgene 12260), and library transfer vectorpLas (Supplementary Sequence 1, lentiviral backbone derived from Addgene61427) was transfected into 293FT cells (Thermo Fisher R70007) usingLipofectamine 2000 (Invitrogen 11668019). Fresh media was exchanged 4 hafter transfection. At 24 h post-transfection, 2 mM caffeine(Sigma-Aldrich C0750) was added, and at 48 h post-transfectionlentiviral supernatant was filtered through 0.45 μm cellulose acetatefilters (VWR 28145-481), frozen at −80° C., and thawed immediatelybefore use. HeLa cells (a gift from Dr. Iain Cheeseman's lab) wereinfected by mixing lentiviral supernatant with 8 μg/mL polybrene(Sigma-Aldrich 107689-10G) and centrifuging at 1000 g for 2 h at 33° C.At 6 h post-infection, media was exchanged, and at 24 h post-infectioncells were passaged into media containing 300 μg/mL zeocin (ThermoFisher R25001) and selected for one week. Single cells were sorted into96-well plates and clonally expanded. More than 90% of wells with cellgrowth contained single colonies, determined by visual inspection. Theseexpanded clones were analyzed by extracting gDNA, separately amplifyingsgRNA and barcode sequences by PCR, and deep sequencing the amplicons(Illumina Mini Seq).

Sequence data from each colony were analyzed by matching reads to knownsgRNAs within an allowed edit distance of 2 bases and barcodes within anallowed edit distance of 1 base to accommodate errors in oligo synthesis(Supplementary Table 1). Sequences with fewer than 30 reads or a readfraction below 10% were discarded. Multiple integration events weredefined by the presence of more than one sgRNA or more than one barcodesequence. Single integration with barcode swapping was defined asdetection of one sgRNA and one barcode cognate to a different sgRNA. Wereport the frequency of observed barcode swapping events, which does notinclude multiple integration of the same library element, recombinationbetween two identical library elements, or secondary recombinationevents that restore the original sgRNA-barcode pairing.

sgRNA vector sequences and vector details are provided below in Table 2.(Pred sgRNA identified as SEQ ID NOs: 1-945) (Required “match” sequencesidentified as SEQ ID NOs: 946-1573)

TABLE 2 mat- ches_ per_ pat- frac- soli- sam- condition date plate welltern tion pred_sgRNA tary ple match count pLas, 20170 T1 B09 pL43 83.80%AGAGCACTGCA FALSE 2 CCAGTTA 3540 individually   608 CTCCTTCA packagedpLas, 20170 T1 B09 sgRNA 92.70% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  356individually   608 CTCCTTCA TCCTTCA packaged pLas, 20170 T1 B12 pL4387.40% AGAGCACTGCA FALSE 2 CCAGTTA 3955 individually   608 CTCCTTCApackaged pLas, 20170 T1 B12 sgRNA 96.00% AGAGCACTGCA FALSE 2AGAGCACTGCAC  924 individually   608 CTCCTTCA TCCTTCA packaged pLas,20170 T1 C09 pL43 95.70% AGTAGTCCGGG FALSE 2 CCTCTTC 3707 individually  608 ATATCAGCG packaged pLas, 20170 T1 C09 sgRNA 95.80% AGTAGTCCGGGFALSE 2 AGTAGTCCGGGA 1451 individually   608 ATATCAGCG TATCAGCG packagedpLas, 20170 T1 C10 pL43 95.70% ATACAACTGCTT FALSE 2 ATTCCGA 4431individually   608 GCAACAGG packaged pLas, 20170 T1 C10 sgRNA 93.60%ATACAACTGCTT FALSE 2 ATACAACTGCTT  904 individually   608 GCAACAGGGCAACAGG packaged pLas, 20170 T1 C12 pL43 94.40% TCCACCGGCGA FALSE 2CAATCGG 4541 individually   608 AAGAGATCC packaged pLas, 20170 T1 C12sgRNA 94.40% TCCACCGGCGA FALSE 2 TCCACCGGCGAA   34 individually   608AAGAGATCC AGAGATCC packaged pLas, 20170 T1 D09 pL43 96.00% CGCCGCCCCCGFALSE 2 CATGCGT 4873 individually   608 GACGCGACC packaged pLas, 20170T1 D09 sgRNA 94.90% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  806 individually  608 GACGCGACC ACGCGACC packaged pLas, 20170 T1 D11 pL43 10.60%TCATATTACGAG TRUE 3 AGAGAGA  493 individually   608 TCAGTAGG packagedpLas, 20170 T1 D11 pL43 85.30% AGAGCACTGCA FALSE 3 CCAGTTA 3980individually   608 CTCCTTCA packaged pLas, 20170 T1 D11 sgRNA 87.60%AGAGCACTGCA FALSE 3 AGAGCACTGCAC  212 individually   608 CTCCTTCATCCTTCA packaged pLas, 20170 T1 D12 pL43 94.80% TCATATTACGAG FALSE 2AGAGAGA 4567 individually   608 TCAGTAGG packaged pLas, 20170 T1 D12sgRNA 94.60% TCATATTACGAG FALSE 2 TCATATTACGAGT 1346 individually   608TCAGTAGG CAGTAGG packaged pLas, 20170 T1 E10 pL43 95.10% CCAGTACAAACFALSE 2 AAGAGGA 4585 individually   608 CTACCTACG packaged pLas, 20170T1 E10 sgRNA 95.70% CCAGTACAAAC FALSE 2 CCAGTACAAACC  425 individually  608 CTACCTACG TACCTACG packaged pLas, 20170 T1 E11 pL43 94.70%AGAGCACTGCA FALSE 2 CCAGTTA 4781 individually   608 CTCCTTCA packagedpLas, 20170 T1 E11 sgRNA 95.50% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  233individually   608 CTCCTTCA TCCTTCA packaged pLas, 20170 T1 E12 pL4394.20% AGAGCACTGCA FALSE 2 CCAGTTA 4257 individually   608 CTCCTTCApackaged pLas, 20170 T1 E12 sgRNA 95.80% AGAGCACTGCA FALSE 2AGAGCACTGCAC 1084 individually   608 CTCCTTCA TCCTTCA packaged pLas,20170 T1 F10 pL43 94.70% AGAGCACTGCA FALSE 2 CCAGTTA 3828 individually  608 CTCCTTCA packaged pLas, 20170 T1 F10 sgRNA 93.30% AGAGCACTGCAFALSE 2 AGAGCACTGCAC  532 individually   608 CTCCTTCA TCCTTCA packagedpLas, 20170 T1 F11 pL43 47.30% CCTGCAACGGG FALSE 4 CGTCATA 2137individually   608 ACTAGTTGG packaged pLas, 20170 T1 F11 pL43 48.40%ATACAACTGCTT FALSE 4 ATTCCGA 2184 individually   608 GCAACAGG packagedpLas, 20170 T1 F11 sgRNA 46.70% ATACAACTGCTT FALSE 4 ATACAACTGCTT  314individually   608 GCAACAGG GCAACAGG packaged pLas, 20170 T1 F11 sgRNA46.80% CCTGCAACGGG FALSE 4 CCTGCAACGGGA  315 individually   608ACTAGTTGG CTAGTTGG packaged pLas, 20170 T1 F12 pL43 95.60% CGCCGCCCCCGFALSE 2 CATGCGT 4409 individually   608 GACGCGACC packaged pLas, 20170T1 F12 sgRNA 95.30% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  843 individually  608 GACGCGACC ACGCGACC packaged pLas, 20170 T1 G09 pL43 79.80%AGAGCACTGCA FALSE 2 CCAGTTA 2743 individually   608 CTCCTTCA packagedpLas, 20170 T1 G09 sgRNA 94.10% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  434individually   608 CTCCTTCA TCCTTCA packaged pLas, 20170 T1 G12 pL4390.80% CCAGTACAAAC FALSE 2 AAGAGGA 4450 individually   608 CTACCTACGpackaged pLas, 20170 T1 G12 sgRNA 94.60% CCAGTACAAAC FALSE 2CCAGTACAAACC  945 individually   608 CTACCTACG TACCTACG packaged pLas,20170 T1 H09 pL43 95.20% CGCCGCCCCCG FALSE 2 CATGCGT 4742 individually  608 GACGCGACC packaged pLas, 20170 T1 H09 sgRNA 95.90% CGCCGCCCCCGFALSE 2 CGCCGCCCCCGG 1368 individually   608 GACGCGACC ACGCGACC packagedpLas, 20170 T1 H10 pL43 95.60% CGCCGCCCCCG FALSE 2 CATGCGT 4531individually   608 GACGCGACC packaged pLas, 20170 T1 H10 sgRNA 95.60%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  900 individually   608 GACGCGACCACGCGACC packaged pLas, 20170 T1 H11 pL43 86.10% TCCACCGGCGA FALSE 2CAATCGG 3570 individually   608 AAGAGATCC packaged pLas, 20170 T1 H11sgRNA 94.80% TCCACCGGCGA FALSE 2 TCCACCGGCGAA 2455 individually   608AAGAGATCC AGAGATCC packaged pLas, 20170 T1 H12 pL43 80.90% ATACAACTGCTTFALSE 2 ATTCCGA 3032 individually   608 GCAACAGG packaged pLas, 20170 T1H12 sgRNA 87.80% ATACAACTGCTT FALSE 2 ATACAACTGCTT 1155 individually  608 GCAACAGG GCAACAGG packaged pLas, 20170 T2 B01 pL43 43.50%AGAGCACTGCA FALSE 4 CCAGTTA 1799 individually   608 CTCCTTCA packagedpLas, 20170 T2 B01 pL43 53.90% TCATATTACGAG FALSE 4 AGAGAGA 2225individually   608 TCAGTAGG packaged pLas, 20170 T2 B01 sgRNA 46.70%AGAGCACTGCA FALSE 4 AGAGCACTGCAC  293 individually   608 CTCCTTCATCCTTCA packaged pLas, 20170 T2 B01 sgRNA 48.70% TCATATTACGAG FALSE 4TCATATTACGAGT  306 individually   608 TCAGTAGG CAGTAGG packaged pLas,20170 T2 B02 pL43 96.60% CGCCGCCCCCG FALSE 2 CATGCGT 4180 individually  608 GACGCGACC packaged pLas, 20170 T2 B02 sgRNA 95.10% CGCCGCCCCCGFALSE 2 CGCCGCCCCCGG  874 individually   608 GACGCGACC ACGCGACC packagedpLas, 20170 T2 B03 pL43 96.90% CCTGCAACGGG FALSE 2 CGTCATA 4136individually   608 ACTAGTTGG packaged pLas, 20170 T2 B03 sgRNA 95.80%CCTGCAACGGG FALSE 2 CCTGCAACGGGA  877 individually   608 ACTAGTTGGCTAGTTGG packaged pLas, 20170 T2 B04 pL43 96.00% AGTAGTCCGGG FALSE 2CCTCTTC 3368 individually   608 ATATCAGCG packaged pLas, 20170 T2 B04sgRNA 93.70% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  388 individually   608ATATCAGCG TATCAGCG packaged pLas, 20170 T2 C01 pL43 96.50% AGTAGTCCGGGFALSE 2 CCTCTTC 3512 individually   608 ATATCAGCG packaged pLas, 20170T2 C01 sgRNA 96.50% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  599 individually  608 ATATCAGCG TATCAGCG packaged pLas, 20170 T2 C02 pL43 94.50%CCTGCAACGGG FALSE 2 CGTCATA 3691 individually   608 ACTAGTTGG packagedpLas, 20170 T2 C02 sgRNA 95.50% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  764individually   608 ACTAGTTGG CTAGTTGG packaged pLas, 20170 T2 C03 pL4396.90% ATACAACTGCTT FALSE 2 ATTCCGA 4307 individually   608 GCAACAGGpackaged pLas, 20170 T2 C03 sgRNA 93.80% ATACAACTGCTT FALSE 2ATACAACTGCTT  405 individually   608 GCAACAGG GCAACAGG packaged pLas,20170 T2 C04 pL43 97.20% CGCCGCCCCCG FALSE 2 CATGCGT 4177 individually  608 GACGCGACC packaged pLas, 20170 T2 C04 sgRNA 96.30% CGCCGCCCCCGFALSE 2 CGCCGCCCCCGG  235 individually   608 GACGCGACC ACGCGACC packagedpLas, 20170 T2 D01 pL43 97.00% TCATATTACGAG FALSE 2 AGAGAGA 3954individually   608 TCAGTAGG packaged pLas, 20170 T2 D01 sgRNA 95.90%TCATATTACGAG FALSE 2 TCATATTACGAGT  352 individually   608 TCAGTAGGCAGTAGG packaged pLas, 20170 T2 D02 pL43 94.30% TCATATTACGAG FALSE 2AGAGAGA 3606 individually   608 TCAGTAGG packaged pLas, 20170 T2 D02sgRNA 96.00% TCATATTACGAG FALSE 2 TCATATTACGAGT 1285 individually   608TCAGTAGG CAGTAGG packaged pLas, 20170 T2 D03 pL43 95.50% CCTGCAACGGGFALSE 2 CGTCATA 3714 individually   608 ACTAGTTGG packaged pLas, 20170T2 D03 sgRNA 94.80% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1102 individually  608 ACTAGTTGG CTAGTTGG packaged pLas, 20170 T2 D04 pL43 94.70%CCAGTACAAAC FALSE 2 AAGAGGA 3834 individually   608 CTACCTACG packagedpLas, 20170 T2 D04 sgRNA 94.00% CCAGTACAAAC FALSE 2 CCAGTACAAACC  299individually   608 CTACCTACG TACCTACG packaged pLas, 20170 T2 E01 pL4397.00% AGTAGTCCGGG FALSE 2 CCTCTTC 3354 individually   608 ATATCAGCGpackaged pLas, 20170 T2 E01 sgRNA 93.60% AGTAGTCCGGG FALSE 2AGTAGTCCGGGA  497 individually   608 ATATCAGCG TATCAGCG packaged pLas,20170 T2 E02 pL43 96.10% AGAGCACTGCA FALSE 2 CCAGTTA 3865 individually  608 CTCCTTCA packaged pLas, 20170 T2 E02 sgRNA 94.60% AGAGCACTGCAFALSE 2 AGAGCACTGCAC  881 individually   608 CTCCTTCA TCCTTCA packagedpLas, 20170 T2 E03 pL43 97.00% TCATATTACGAG FALSE 2 AGAGAGA 3574individually   608 TCAGTAGG packaged pLas, 20170 T2 E03 sgRNA 95.00%TCATATTACGAG FALSE 2 TCATATTACGAGT  498 individually   608 TCAGTAGGCAGTAGG packaged pLas, 20170 T2 E04 pL43 96.50% AGAGCACTGCA FALSE 2CCAGTTA 4290 individually   608 CTCCTTCA packaged pLas, 20170 T2 E04sgRNA 95.30% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  505 individually   608CTCCTTCA TCCTTCA packaged pLas, 20170 T2 F01 pL43 96.60% TCCACCGGCGAFALSE 2 CAATCGG 4255 individually   608 AAGAGATCC packaged pLas, 20170T2 F01 sgRNA 94.60% TCCACCGGCGA FALSE 2 TCCACCGGCGAA  905 individually  608 AAGAGATCC AGAGATCC packaged pLas, 20170 T2 F02 pL43 97.30%ATACAACTGCTT FALSE 2 ATTCCGA 4292 individually   608 GCAACAGG packagedpLas, 20170 T2 F02 sgRNA 96.50% ATACAACTGCTT FALSE 2 ATACAACTGCTT  625individually   608 GCAACAGG GCAACAGG packaged pLas, 20170 T2 F03 pL4397.40% CCTGCAACGGG FALSE 2 CGTCATA 4249 individually   608 ACTAGTTGGpackaged pLas, 20170 T2 F03 sgRNA 93.70% CCTGCAACGGG FALSE 2CCTGCAACGGGA  328 individually   608 ACTAGTTGG CTAGTTGG packaged pLas,20170 T2 F04 pL43 96.90% ATACAACTGCTT FALSE 2 ATTCCGA 3725 individually  608 GCAACAGG packaged pLas, 20170 T2 F04 sgRNA 95.10% ATACAACTGCTTFALSE 2 ATACAACTGCTT  254 individually   608 GCAACAGG GCAACAGG packagedpLas, 20170 T2 G01 pL43 97.20% ATACAACTGCTT FALSE 2 ATTCCGA 4241individually   608 GCAACAGG packaged pLas, 20170 T2 G01 sgRNA 95.10%ATACAACTGCTT FALSE 2 ATACAACTGCTT 1193 individually   608 GCAACAGGGCAACAGG packaged pLas, 20170 T2 G02 pL43 97.60% CCTGCAACGGG FALSE 2CGTCATA 4452 individually   608 ACTAGTTGG packaged pLas, 20170 T2 G02sgRNA 94.60% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  712 individually   608ACTAGTTGG CTAGTTGG packaged pLas, 20170 T2 G03 pL43 97.50% TCATATTACGAGFALSE 2 AGAGAGA 4061 individually   608 TCAGTAGG packaged pLas, 20170 T2G03 sgRNA 95.40% TCATATTACGAG FALSE 2 TCATATTACGAGT  536 individually  608 TCAGTAGG CAGTAGG packaged pLas, 20170 T2 G04 pL43 96.90%CGCCGCCCCCG FALSE 2 CATGCGT 3959 individually   608 GACGCGACC packagedpLas, 20170 T2 G04 sgRNA 96.20% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  179individually   608 GACGCGACC ACGCGACC packaged pLas, 20170 T2 H01 pL4397.20% AGAGCACTGCA FALSE 2 CCAGTTA 4232 individually   608 CTCCTTCApackaged pLas, 20170 T2 H01 sgRNA 95.20% AGAGCACTGCA FALSE 2AGAGCACTGCAC 1226 individually   608 CTCCTTCA TCCTTCA packaged pLas,20170 T2 H02 pL43 96.30% AGTAGTCCGGG FALSE 2 CCTCTTC 3553 individually  608 ATATCAGCG packaged pLas, 20170 T2 H02 sgRNA 95.80% AGTAGTCCGGGFALSE 2 AGTAGTCCGGGA 1508 individually   608 ATATCAGCG TATCAGCG packagedpLas, 20170 T2 H03 pL43 97.40% CCAGTACAAAC FALSE 2 AAGAGGA 4423individually   608 CTACCTACG packaged pLas, 20170 T2 H03 sgRNA 96.60%CCAGTACAAAC FALSE 2 CCAGTACAAACC 1501 individually   608 CTACCTACGTACCTACG packaged pLas, 20170 T2 H04 pL43 97.70% CCTGCAACGGG FALSE 2CGTCATA 4444 individually   608 ACTAGTTGG packaged pLas, 20170 T2 H04sgRNA 96.40% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  862 individually   608ACTAGTTGG CTAGTTGG packaged pLas, 20170 T3 A01 pL43 38.30% AGTAGTCCGGGFALSE 5 CCTCTTC  916 standard_8   608 ATATCAGCG pLas, 20170 T3 A01 pL4351.20% ATACAACTGCTT FALSE 5 ATTCCGA 1222 standard_8   608 GCAACAGG pLas,20170 T3 A01 sgRNA 14.30% AGAGCACTGCA TRUE 5 AGAGCACTGCAC  314standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 A01 sgRNA 29.50%AGTAGTCCGGG FALSE 5 AGTAGTCCGGGA  648 standard_8   608 ATATCAGCGTATCAGCG pLas, 20170 T3 A01 sgRNA 49.80% ATACAACTGCTT FALSE 5ATACAACTGCTT 1094 standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 A02pL43 97.10% ATACAACTGCTT FALSE 2 ATTCCGA 4458 standard_8   608 GCAACAGGpLas, 20170 T3 A02 sgRNA 95.20% ATACAACTGCTT FALSE 2 ATACAACTGCTT 1199standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 A03 pL43 96.70%CCTGCAACGGG FALSE 2 CGTCATA 3846 standard_8   608 ACTAGTTGG pLas, 20170T3 A03 sgRNA 96.00% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 3791 standard_8  608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 A04 pL43 12.90% AGAGCACTGCA TRUE3 AGGCTCT  202 standard_8   608 CTCCTTCA pLas, 20170 T3 A04 pL43 75.50%AGAGCACTGCA TRUE 3 CCAGTTA 1184 standard_8   608 CTCCTTCA pLas, 20170 T3A04 sgRNA 95.50% TCATATTACGAG TRUE 3 TCATATTACGAGT 3631 standard_8   608TCAGTAGG CAGTAGG pLas, 20170 T3 A05 pL43 94.30% CGCCGCCCCCG FALSE 2CATGCGT 3658 standard_8   608 GACGCGACC pLas, 20170 T3 A05 sgRNA 92.60%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  312 standard_8   608 GACGCGACCACGCGACC pLas, 20170 T3 A06 pL43 22.20% CCTGCAACGGG TRUE 5 CGTCATA 1003standard_8   608 ACTAGTTGG pLas, 20170 T3 A06 pL43 23.50% ATACAACTGCTTFALSE 5 ATTCCGA 1062 standard_8   608 GCAACAGG pLas, 20170 T3 A06 pL4351.50% TCCACCGGCGA FALSE 5 CAATCGG 2328 standard_8   608 AAGAGATCC pLas,20170 T3 A06 sgRNA 37.00% ATACAACTGCTT FALSE 5 ATACAACTGCTT  125standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 A06 sgRNA 57.40%TCCACCGGCGA FALSE 5 TCCACCGGCGAA  194 standard_8   608 AAGAGATCCAGAGATCC pLas, 20170 T3 A07 pL43 97.60% CCTGCAACGGG TRUE 2 CGTCATA 3824standard_8   608 ACTAGTTGG pLas, 20170 T3 A07 sgRNA 95.00% AGTAGTCCGGGTRUE 2 AGTAGTCCGGGA 1558 standard_8   608 ATATCAGCG TATCAGCG pLas, 20170T3 A08 pL43 91.30% ATACAACTGCTT FALSE 2 ATTCCGA 2460 standard_8   608GCAACAGG pLas, 20170 T3 A08 sgRNA 91.30% ATACAACTGCTT FALSE 2ATACAACTGCTT 2853 standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 B01pL43 91.50% AGTAGTCCGGG FALSE 2 CCTCTTC 2732 standard_8   608 ATATCAGCGpLas, 20170 T3 B01 sgRNA 95.70% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 3419standard_8   608 ATATCAGCG TATCAGCG pLas, 20170 T3 B02 pL43 97.30%CCAGTACAAAC FALSE 2 AAGAGGA 4877 standard_8   608 CTACCTACG pLas, 20170T3 B02 sgRNA 96.20% CCAGTACAAAC FALSE 2 CCAGTACAAACC  403 standard_8  608 CTACCTACG TACCTACG pLas, 20170 T3 B03 pL43 67.90% TCCACCGGCGA TRUE2 CAATCGG 1752 standard_8   608 AAGAGATCC pLas, 20170 T3 B03 sgRNA93.20% ATACAACTGCTT TRUE 2 ATACAACTGCTT  743 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 B04 pL43 96.50% ATACAACTGCTT FALSE 2 ATTCCGA4275 standard_8   608 GCAACAGG pLas, 20170 T3 B04 sgRNA 95.10%ATACAACTGCTT FALSE 2 ATACAACTGCTT  447 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 B05 pL43 97.00% AGTAGTCCGGG FALSE 2 CCTCTTC 3946standard_8   608 ATATCAGCG pLas, 20170 T3 B05 sgRNA 96.40% AGTAGTCCGGGFALSE 2 AGTAGTCCGGGA  986 standard_8   608 ATATCAGCG TATCAGCG pLas,20170 T3 B06 pL43 96.50% AGTAGTCCGGG TRUE 2 CCTCTTC 3955 standard_8  608 ATATCAGCG pLas, 20170 T3 B06 sgRNA 95.30% ATACAACTGCTT TRUE 2ATACAACTGCTT 1075 standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 B07pL43 96.50% CGCCGCCCCCG FALSE 2 CATGCGT 4415 standard_8   608 GACGCGACCpLas, 20170 T3 B07 sgRNA 96.70% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  652standard_8   608 GACGCGACC ACGCGACC pLas, 20170 T3 B08 pL43 45.70%AGTAGTCCGGG FALSE 4 CCTCTTC 1938 standard_8   608 ATATCAGCG pLas, 20170T3 B08 pL43 51.40% AGAGCACTGCA FALSE 4 CCAGTTA 2177 standard_8   608CTCCTTCA pLas, 20170 T3 B08 sgRNA 47.90% AGAGCACTGCA FALSE 4AGAGCACTGCAC  512 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 B08sgRNA 47.90% AGTAGTCCGGG FALSE 4 AGTAGTCCGGGA  513 standard_8   608ATATCAGCG TATCAGCG pLas, 20170 T3 C01 pL43 97.10% ATACAACTGCTT FALSE 2ATTCCGA 5003 standard_8   608 GCAACAGG pLas, 20170 T3 C01 sgRNA 96.40%ATACAACTGCTT FALSE 2 ATACAACTGCTT  782 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 C02 pL43 95.60% CCAGTACAAAC TRUE 2 AAGAGGA 4832standard_8   608 CTACCTACG pLas, 20170 T3 C02 sgRNA 95.90% CCTGCAACGGGTRUE 2 CCTGCAACGGGA 1433 standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170T3 C03 pL43 96.80% CCTGCAACGGG TRUE 2 CGTCATA 5059 standard_8   608ACTAGTTGG pLas, 20170 T3 C03 sgRNA 95.40% AGTAGTCCGGG TRUE 2AGTAGTCCGGGA  638 standard_8   608 ATATCAGCG TATCAGCG pLas, 20170 T3 C04pL43 97.20% CCTGCAACGGG TRUE 2 CGTCATA 4324 standard_8   608 ACTAGTTGGpLas, 20170 T3 C04 sgRNA 95.30% TCATATTACGAG TRUE 2 TCATATTACGAGT  323standard_8   608 TCAGTAGG CAGTAGG pLas, 20170 T3 C05 pL43 97.10%TCCACCGGCGA FALSE 2 CAATCGG 4711 standard_8   608 AAGAGATCC pLas, 20170T3 C05 sgRNA 94.40% TCCACCGGCGA FALSE 2 TCCACCGGCGAA 1218 standard_8  608 AAGAGATCC AGAGATCC pLas, 20170 T3 C06 pL43 94.60% TCATATTACGAGFALSE 2 AGAGAGA 4576 standard_8   608 TCAGTAGG pLas, 20170 T3 C06 sgRNA96.30% TCATATTACGAG FALSE 2 TCATATTACGAGT  949 standard_8   608 TCAGTAGGCAGTAGG pLas, 20170 T3 C07 pL43 93.00% CCAGTACAAAC FALSE 2 AAGAGGA 3743standard_8   608 CTACCTACG pLas, 20170 T3 C07 sgRNA 95.20% CCAGTACAAACFALSE 2 CCAGTACAAACC  758 standard_8   608 CTACCTACG TACCTACG pLas,20170 T3 C08 pL43 95.40% CCAGTACAAAC TRUE 2 AAGAGGA 4504 standard_8  608 CTACCTACG pLas, 20170 T3 C08 sgRNA 95.40% CCTGCAACGGG TRUE 2CCTGCAACGGGA  577 standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 D01pL43 96.00% CCTGCAACGGG FALSE 2 CGTCATA 4673 standard_8   608 ACTAGTTGGpLas, 20170 T3 D01 sgRNA 94.90% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  691standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 D02 pL43 96.20%AGAGCACTGCA FALSE 2 CCAGTTA 5213 standard_8   608 CTCCTTCA pLas, 20170T3 D02 sgRNA 96.10% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  724 standard_8  608 CTCCTTCA TCCTTCA pLas, 20170 T3 D03 pL43 59.90% TCATATTACGAG FALSE2 AGAGAGA 1526 standard_8   608 TCAGTAGG pLas, 20170 T3 D03 sgRNA 94.70%TCATATTACGAG FALSE 2 TCATATTACGAGT  622 standard_8   608 TCAGTAGGCAGTAGG pLas, 20170 T3 D04 pL43 95.00% TCCACCGGCGA FALSE 2 CAATCGG 4568standard_8   608 AAGAGATCC pLas, 20170 T3 D04 sgRNA 95.20% TCCACCGGCGAFALSE 2 TCCACCGGCGAA  239 standard_8   608 AAGAGATCC AGAGATCC pLas,20170 T3 D05 pL43 96.50% CGCCGCCCCCG FALSE 2 CATGCGT 4945 standard_8  608 GACGCGACC pLas, 20170 T3 D05 sgRNA 96.90% CGCCGCCCCCG FALSE 2CGCCGCCCCCGG  409 standard_8   608 GACGCGACC ACGCGACC pLas, 20170 T3 D06pL43 96.50% ATACAACTGCTT FALSE 2 ATTCCGA 4165 standard_8   608 GCAACAGGpLas, 20170 T3 D06 sgRNA 94.60% ATACAACTGCTT FALSE 2 ATACAACTGCTT  591standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 D07 pL43 96.80%CCAGTACAAAC FALSE 2 AAGAGGA 4170 standard_8   608 CTACCTACG pLas, 20170T3 D07 sgRNA 96.80% CCAGTACAAAC FALSE 2 CCAGTACAAACC  605 standard_8  608 CTACCTACG TACCTACG pLas, 20170 T3 D08 pL43 48.30% CCTGCAACGGGFALSE 4 CGTCATA 2156 standard_8   608 ACTAGTTGG pLas, 20170 T3 D08 pL4348.90% TCATATTACGAG FALSE 4 AGAGAGA 2180 standard_8   608 TCAGTAGG pLas,20170 T3 D08 sgRNA 45.20% TCATATTACGAG FALSE 4 TCATATTACGAGT  150standard_8   608 TCAGTAGG CAGTAGG pLas, 20170 T3 D08 sgRNA 49.40%CCTGCAACGGG FALSE 4 CCTGCAACGGGA  164 standard_8   608 ACTAGTTGGCTAGTTGG pLas, 20170 T3 E01 pL43 93.70% AGAGCACTGCA FALSE 2 CCAGTTA 3427standard_8   608 CTCCTTCA pLas, 20170 T3 E01 sgRNA 94.20% AGAGCACTGCAFALSE 2 AGAGCACTGCAC 1096 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170T3 E02 pL43 44.00% AGAGCACTGCA FALSE 4 CCAGTTA 1441 standard_8   608CTCCTTCA pLas, 20170 T3 E02 pL43 52.70% ATACAACTGCTT FALSE 4 ATTCCGA1726 standard_8   608 GCAACAGG pLas, 20170 T3 E02 sgRNA 45.10%ATACAACTGCTT FALSE 4 ATACAACTGCTT  208 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 E02 sgRNA 48.60% AGAGCACTGCA FALSE 4AGAGCACTGCAC  224 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 E03pL43 97.40% CCTGCAACGGG FALSE 2 CGTCATA 3657 standard_8   608 ACTAGTTGGpLas, 20170 T3 E03 sgRNA 95.50% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  385standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 E04 pL43 97.20%TCATATTACGAG FALSE 2 AGAGAGA 3853 standard_8   608 TCAGTAGG pLas, 20170T3 E04 sgRNA 95.10% TCATATTACGAG FALSE 2 TCATATTACGAGT  254 standard_8  608 TCAGTAGG CAGTAGG pLas, 20170 T3 E05 pL43 96.60% CGCCGCCCCCG FALSE2 CATGCGT 3585 standard_8   608 GACGCGACC pLas, 20170 T3 E05 sgRNA97.30% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  655 standard_8   608 GACGCGACCACGCGACC pLas, 20170 T3 E06 pL43 97.60% TCATATTACGAG TRUE 2 AGAGAGA 3875standard_8   608 TCAGTAGG pLas, 20170 T3 E06 sgRNA 92.90% ATACAACTGCTTTRUE 2 ATACAACTGCTT  262 standard_8   608 GCAACAGG GCAACAGG pLas, 20170T3 E07 pL43 97.10% ATACAACTGCTT TRUE 2 ATTCCGA 3665 standard_8   608GCAACAGG pLas, 20170 T3 E07 sgRNA 94.50% AGAGCACTGCA TRUE 2 AGAGCACTGCAC 241 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 E08 pL43 95.40%AGAGCACTGCA FALSE 2 CCAGTTA 3312 standard_8   608 CTCCTTCA pLas, 20170T3 E08 sgRNA 95.40% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  248 standard_8  608 CTCCTTCA TCCTTCA pLas, 20170 T3 F01 pL43 97.10% CCAGTACAAAC TRUE 2AAGAGGA 4357 standard_8   608 CTACCTACG pLas, 20170 T3 F01 sgRNA 94.70%AGAGCACTGCA TRUE 2 AGAGCACTGCAC 1724 standard_8   608 CTCCTTCA TCCTTCApLas, 20170 T3 F03 pL43 97.00% ATACAACTGCTT FALSE 2 ATTCCGA 4167standard_8   608 GCAACAGG pLas, 20170 T3 F03 sgRNA 95.40% ATACAACTGCTTFALSE 2 ATACAACTGCTT  740 standard_8   608 GCAACAGG GCAACAGG pLas, 20170T3 F04 pL43 96.70% AGAGCACTGCA TRUE 2 CCAGTTA 4419 standard_8   608CTCCTTCA pLas, 20170 T3 F04 sgRNA 96.20% CCTGCAACGGG TRUE 2 CCTGCAACGGGA 332 standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 F05 pL43 96.70%AGAGCACTGCA TRUE 2 CCAGTTA 4215 standard_8   608 CTCCTTCA pLas, 20170 T3F05 sgRNA 96.20% CCTGCAACGGG TRUE 2 CCTGCAACGGGA  833 standard_8   608ACTAGTTGG CTAGTTGG pLas, 20170 T3 F06 pL43 96.80% CGCCGCCCCCG TRUE 2CATGCGT 3523 standard_8   608 GACGCGACC pLas, 20170 T3 F06 sgRNA 95.50%CCTGCAACGGG TRUE 2 CCTGCAACGGGA  231 standard_8   608 ACTAGTTGG CTAGTTGGpLas, 20170 T3 F07 pL43 95.90% AGAGCACTGCA FALSE 2 CCAGTTA 3831standard_8   608 CTCCTTCA pLas, 20170 T3 F07 sgRNA 96.00% AGAGCACTGCAFALSE 2 AGAGCACTGCAC  697 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170T3 F08 pL43 96.50% AGAGCACTGCA FALSE 2 CCAGTTA 3858 standard_8   608CTCCTTCA pLas, 20170 T3 F08 sgRNA 94.60% AGAGCACTGCA FALSE 2AGAGCACTGCAC 1214 standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 G01pL43 96.80% TCATATTACGAG FALSE 2 AGAGAGA 4622 standard_8   608 TCAGTAGGpLas, 20170 T3 G01 sgRNA 94.10% TCATATTACGAG FALSE 2 TCATATTACGAGT 1423standard_8   608 TCAGTAGG CAGTAGG pLas, 20170 T3 G02 pL43 46.40%CCAGTACAAAC FALSE 3 AAGAGGA 2320 standard_8   608 CTACCTACG pLas, 20170T3 G02 pL43 51.00% TCATATTACGAG TRUE 3 AGAGAGA 2550 standard_8   608TCAGTAGG pLas, 20170 T3 G02 sgRNA 95.50% CCAGTACAAAC FALSE 3CCAGTACAAACC  612 standard_8   608 CTACCTACG TACCTACG pLas, 20170 T3 G05pL43 96.60% CCTGCAACGGG FALSE 2 CGTCATA 3919 standard_8   608 ACTAGTTGGpLas, 20170 T3 G05 sgRNA 95.80% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 2747standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170 T3 G06 pL43 95.30%ATACAACTGCTT TRUE 2 ATTCCGA 4219 standard_8   608 GCAACAGG pLas, 20170T3 G06 sgRNA 94.70% AGAGCACTGCA TRUE 2 AGAGCACTGCAC 1060 standard_8  608 CTCCTTCA TCCTTCA pLas, 20170 T3 G07 pL43 96.90% TCATATTACGAG FALSE2 AGAGAGA 4940 standard_8   608 TCAGTAGG pLas, 20170 T3 G07 sgRNA 94.90%TCATATTACGAG FALSE 2 TCATATTACGAGT  991 standard_8   608 TCAGTAGGCAGTAGG pLas, 20170 T3 G08 pL43 97.50% TCATATTACGAG FALSE 2 AGAGAGA 4771standard_8   608 TCAGTAGG pLas, 20170 T3 G08 sgRNA 95.50% TCATATTACGAGFALSE 2 TCATATTACGAGT 1030 standard_8   608 TCAGTAGG CAGTAGG pLas, 20170T3 H01 pL43 97.10% CCTGCAACGGG TRUE 2 CGTCATA 4412 standard_8   608ACTAGTTGG pLas, 20170 T3 H01 sgRNA 95.00% ATACAACTGCTT TRUE 2ATACAACTGCTT  830 standard_8   608 GCAACAGG GCAACAGG pLas, 20170 T3 H02pL43 96.60% ATACAACTGCTT TRUE 2 ATTCCGA 4004 standard_8   608 GCAACAGGpLas, 20170 T3 H02 sgRNA 95.00% AGAGCACTGCA TRUE 2 AGAGCACTGCAC 1371standard_8   608 CTCCTTCA TCCTTCA pLas, 20170 T3 H03 pL43 96.90%TCCACCGGCGA TRUE 2 CAATCGG 3486 standard_8   608 AAGAGATCC pLas, 20170T3 H03 sgRNA 92.90% ATACAACTGCTT TRUE 2 ATACAACTGCTT   39 standard_8  608 GCAACAGG GCAACAGG pLas, 20170 T3 H04 pL43 97.80% ATACAACTGCTTFALSE 2 ATTCCGA 4240 standard_8   608 GCAACAGG pLas, 20170 T3 H04 sgRNA94.60% ATACAACTGCTT FALSE 2 ATACAACTGCTT  227 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 H05 pL43 97.30% TCATATTACGAG TRUE 2 AGAGAGA 4390standard_8   608 TCAGTAGG pLas, 20170 T3 H05 sgRNA 95.90% CCTGCAACGGGTRUE 2 CCTGCAACGGGA  375 standard_8   608 ACTAGTTGG CTAGTTGG pLas, 20170T3 H06 pL43 30.90% AGAGCACTGCA TRUE 3 AGGCTCT  715 standard_8   608CTCCTTCA pLas, 20170 T3 H06 pL43 62.70% ATACAACTGCTT FALSE 3 ATTCCGA1451 standard_8   608 GCAACAGG pLas, 20170 T3 H06 sgRNA 95.00%ATACAACTGCTT FALSE 3 ATACAACTGCTT 3966 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 H07 pL43 97.30% ATACAACTGCTT FALSE 2 ATTCCGA4431 standard_8   608 GCAACAGG pLas, 20170 T3 H07 sgRNA 95.00%ATACAACTGCTT FALSE 2 ATACAACTGCTT  707 standard_8   608 GCAACAGGGCAACAGG pLas, 20170 T3 H08 pL43 97.60% AGAGCACTGCA TRUE 2 CCAGTTA 4408standard_8   608 CTCCTTCA pLas, 20170 T3 H08 sgRNA 94.20% ATACAACTGCTTTRUE 2 ATACAACTGCTT 1007 standard_8   608 GCAACAGG GCAACAGG pLas + 20170T4 A05 pL43 97.50% AGAGCACTGCA FALSE 2 CCAGTTA 4557 pR_H2B-BFP   608CTCCTTCA (1:1000) pLas + 20170 T4 A05 sgRNA 94.40% AGAGCACTGCA FALSE 2AGAGCACTGCAC 1303 pR_H2B-BFP   608 CTCCTTCA TCCTTCA (1:1000) pLas +20170 T4 A06 pL43 97.40% CGCCGCCCCCG FALSE 2 CATGCGT 4231 pR_H2B-BFP  608 GACGCGACC (1:1000) pLas + 20170 T4 A06 sgRNA 96.40% CGCCGCCCCCGFALSE 2 CGCCGCCCCCGG 1004 pR_H2B-BFP   608 GACGCGACC ACGCGACC (1:1000)pLas + 20170 T4 A07 pL43 97.00% AGAGCACTGCA FALSE 2 CCAGTTA 4116pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4 A07 sgRNA 94.60%AGAGCACTGCA FALSE 2 AGAGCACTGCAC  823 pR_H2B-BFP   608 CTCCTTCA TCCTTCA(1:1000) pLas + 20170 T4 A08 pL43 97.60% TCATATTACGAG FALSE 2 AGAGAGA3955 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4 A08 sgRNA 90.90%TCATATTACGAG FALSE 2 TCATATTACGAGT  765 pR_H2B-BFP   608 TCAGTAGGCAGTAGG (1:1000) pLas + 20170 T4 A09 pL43 93.70% CCAGTACAAAC FALSE 2AAGAGGA 3001 pR_H2B-BFP   608 CTACCTACG (1:1000) pLas + 20170 T4 A09sgRNA 95.60% CCAGTACAAAC FALSE 2 CCAGTACAAACC 5907 pR_H2B-BFP   608CTACCTACG TACCTACG (1:1000) pLas + 20170 T4 A10 pL43 96.70% AGAGCACTGCAFALSE 2 CCAGTTA 4527 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4A10 sgRNA 95.00% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  906 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 A11 pL43 97.40% CCTGCAACGGGFALSE 2 CGTCATA 4269 pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4A11 sgRNA 96.30% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1234 pR_H2B-BFP   608ACTAGTTGG CTAGTTGG (1:1000) pLas + 20170 T4 A12 pL43 92.50% CCTGCAACGGGFALSE 2 CGTCATA 3147 pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4A12 sgRNA 94.80% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 3223 pR_H2B-BFP   608ACTAGTTGG CTAGTTGG (1:1000) pLas + 20170 T4 B05 pL43 97.20% TCATATTACGAGFALSE 2 AGAGAGA 3990 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4B05 sgRNA 95.70% TCATATTACGAG FALSE 2 TCATATTACGAGT  902 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 B06 pL43 95.80%TCCACCGGCGA FALSE 2 CAATCGG 3985 pR_H2B-BFP   608 AAGAGATCC (1:1000)pLas + 20170 T4 B06 sgRNA 94.60% TCCACCGGCGA FALSE 2 TCCACCGGCGAA  821pR_H2B-BFP   608 AAGAGATCC AGAGATCC (1:1000) pLas + 20170 T4 B07 pL4394.00% AGTAGTCCGGG FALSE 2 CCTCTTC 3237 pR_H2B-BFP   608 ATATCAGCG(1:1000) pLas + 20170 T4 B07 sgRNA 95.40% AGTAGTCCGGG FALSE 2AGTAGTCCGGGA 2166 pR_H2B-BFP   608 ATATCAGCG TATCAGCG (1:1000) pLas +20170 T4 B08 pL43 96.20% CCAGTACAAAC FALSE 2 AAGAGGA 4316 pR_H2B-BFP  608 CTACCTACG (1:1000) pLas + 20170 T4 B08 sgRNA 94.40% CCAGTACAAACFALSE 2 CCAGTACAAACC 1056 pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000)pLas + 20170 T4 B09 pL43 68.70% CCTGCAACGGG FALSE 2 CGTCATA 2349pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4 B09 sgRNA 94.90%CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1363 pR_H2B-BFP   608 ACTAGTTGGCTAGTTGG (1:1000) pLas + 20170 T4 B10 pL43 90.00% TCATATTACGAG FALSE 2AGAGAGA 3146 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4 B10sgRNA 94.90% TCATATTACGAG FALSE 2 TCATATTACGAGT  259 pR_H2B-BFP   608TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 B11 pL43 90.00% AGAGCACTGCAFALSE 2 CCAGTTA 3920 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4B11 sgRNA 95.10% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  409 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 B12 pL43 79.40% CGCCGCCCCCGFALSE 2 CATGCGT 3303 pR_H2B-BFP   608 GACGCGACC (1:1000) pLas + 20170 T4B12 sgRNA 93.20% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  547 pR_H2B-BFP   608GACGCGACC ACGCGACC (1:1000) pLas + 20170 T4 C05 pL43 96.40% AGAGCACTGCAFALSE 2 CCAGTTA 3254 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4C05 sgRNA 94.70% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1888 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 C06 pL43 96.40% AGTAGTCCGGGFALSE 2 CCTCTTC 3564 pR_H2B-BFP   608 ATATCAGCG (1:1000) pLas + 20170 T4C06 sgRNA 96.70% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  231 pR_H2B-BFP   608ATATCAGCG TATCAGCG (1:1000) pLas + 20170 T4 C07 pL43 96.30% AGAGCACTGCAFALSE 2 CCAGTTA 4010 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4C07 sgRNA 95.30% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1061 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 C08 pL43 93.40% ATACAACTGCTTFALSE 2 ATTCCGA 3041 pR_H2B-BFP   608 GCAACAGG (1:1000) pLas + 20170 T4C08 sgRNA 94.70% ATACAACTGCTT FALSE 2 ATACAACTGCTT  537 pR_H2B-BFP   608GCAACAGG GCAACAGG (1:1000) pLas + 20170 T4 C09 pL43 94.60% AGAGCACTGCAFALSE 2 CCAGTTA 3373 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4C09 sgRNA 94.70% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1522 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 C10 pL43 94.70% TCCACCGGCGAFALSE 2 CAATCGG 3893 pR_H2B-BFP   608 AAGAGATCC (1:1000) pLas + 20170 T4C10 sgRNA 92.90% TCCACCGGCGA FALSE 2 TCCACCGGCGAA 1347 pR_H2B-BFP   608AAGAGATCC AGAGATCC (1:1000) pLas + 20170 T4 C11 pL43 83.40% AGTAGTCCGGGFALSE 2 CCTCTTC 2594 pR_H2B-BFP   608 ATATCAGCG (1:1000) pLas + 20170 T4C11 sgRNA 95.80% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 1086 pR_H2B-BFP   608ATATCAGCG TATCAGCG (1:1000) pLas + 20170 T4 C12 pL43 92.90% TCATATTACGAGFALSE 2 AGAGAGA 3721 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4C12 sgRNA 95.90% TCATATTACGAG FALSE 2 TCATATTACGAGT  635 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 D05 pL43 96.20%AGAGCACTGCA FALSE 2 CCAGTTA 3267 pR_H2B-BFP   608 CTCCTTCA (1:1000)pLas + 20170 T4 D05 sgRNA 93.90% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  962pR_H2B-BFP   608 CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 D06 pL4396.80% CCAGTACAAAC FALSE 2 AAGAGGA 4031 pR_H2B-BFP   608 CTACCTACG(1:1000) pLas + 20170 T4 D06 sgRNA 96.20% CCAGTACAAAC FALSE 2CCAGTACAAACC  410 pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000) pLas +20170 T4 D07 pL43 97.00% CCAGTACAAAC FALSE 2 AAGAGGA 3789 pR_H2B-BFP  608 CTACCTACG (1:1000) pLas + 20170 T4 D07 sgRNA 96.70% CCAGTACAAACFALSE 2 CCAGTACAAACC  232 pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000)pLas + 20170 T4 D08 pL43 94.40% AGAGCACTGCA FALSE 2 CCAGTTA 3480pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4 D08 sgRNA 94.80%AGAGCACTGCA FALSE 2 AGAGCACTGCAC  921 pR_H2B-BFP   608 CTCCTTCA TCCTTCA(1:1000) pLas + 20170 T4 D09 pL43 95.90% ATACAACTGCTT FALSE 2 ATTCCGA3652 pR_H2B-BFP   608 GCAACAGG (1:1000) pLas + 20170 T4 D09 sgRNA 95.10%ATACAACTGCTT FALSE 2 ATACAACTGCTT  501 pR_H2B-BFP   608 GCAACAGGGCAACAGG (1:1000) pLas + 20170 T4 D10 pL43 85.60% AGAGCACTGCA FALSE 2CCAGTTA 2837 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4 D10sgRNA 94.20% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 3426 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 D11 pL43 11.50% TCCACCGGCGATRUE 6 CAATCGG  259 pR_H2B-BFP   608 AAGAGATCC (1:1000) pLas + 20170 T4D11 pL43 12.70% AGAGCACTGCA FALSE 6 AGGCTCT  285 pR_H2B-BFP   608CTCCTTCA (1:1000) pLas + 20170 T4 D11 pL43 17.10% AGAGCACTGCA FALSE 6CCAGTTA  385 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4 D11 pL4319.90% CCAGTACAAAC TRUE 6 AAGAGGA  447 pR_H2B-BFP   608 CTACCTACG(1:1000) pLas + 20170 T4 D11 pL43 27.90% TCATATTACGAG TRUE 6 AGAGAGA 628 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4 D11 sgRNA 94.40%AGAGCACTGCA FALSE 6 AGAGCACTGCAC 4967 pR_H2B-BFP   608 CTCCTTCA TCCTTCA(1:1000) pLas + 20170 T4 D12 pL43 91.10% CGCCGCCCCCG FALSE 2 CATGCGT3688 pR_H2B-BFP   608 GACGCGACC (1:1000) pLas + 20170 T4 D12 sgRNA96.30% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  678 pR_H2B-BFP   608 GACGCGACCACGCGACC (1:1000) pLas + 20170 T4 E05 pL43 96.60% AGTAGTCCGGG FALSE 2CCTCTTC 3289 pR_H2B-BFP   608 ATATCAGCG (1:1000) pLas + 20170 T4 E05sgRNA 95.60% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 1545 pR_H2B-BFP   608ATATCAGCG TATCAGCG (1:1000) pLas + 20170 T4 E06 pL43 97.10% ATACAACTGCTTFALSE 2 ATTCCGA 4055 pR_H2B-BFP   608 GCAACAGG (1:1000) pLas + 20170 T4E06 sgRNA 95.30% ATACAACTGCTT FALSE 2 ATACAACTGCTT  763 pR_H2B-BFP   608GCAACAGG GCAACAGG (1:1000) pLas + 20170 T4 E07 pL43 97.00% TCCACCGGCGAFALSE 2 CAATCGG 4106 pR_H2B-BFP   608 AAGAGATCC (1:1000) pLas + 20170 T4E07 sgRNA 93.70% TCCACCGGCGA FALSE 2 TCCACCGGCGAA  298 pR_H2B-BFP   608AAGAGATCC AGAGATCC (1:1000) pLas + 20170 T4 E08 pL43 95.50% TCATATTACGAGFALSE 2 AGAGAGA 4002 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4E08 sgRNA 98.30% TCATATTACGAG FALSE 2 TCATATTACGAGT  116 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 E09 pL43 89.30%TCATATTACGAG FALSE 2 AGAGAGA 2769 pR_H2B-BFP   608 TCAGTAGG (1:1000)pLas + 20170 T4 E09 sgRNA 95.10% TCATATTACGAG FALSE 2 TCATATTACGAGT  741pR_H2B-BFP   608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 E10 pL4388.50% CCTGCAACGGG FALSE 2 CGTCATA 3852 pR_H2B-BFP   608 ACTAGTTGG(1:1000) pLas + 20170 T4 E10 sgRNA 96.00% CCTGCAACGGG FALSE 2CCTGCAACGGGA  819 pR_H2B-BFP   608 ACTAGTTGG CTAGTTGG (1:1000) pLas +20170 T4 E11 pL43 89.60% AGTAGTCCGGG FALSE 2 CCTCTTC 3194 pR_H2B-BFP  608 ATATCAGCG (1:1000) pLas + 20170 T4 E11 sgRNA 97.00% AGTAGTCCGGGFALSE 2 AGTAGTCCGGGA  292 pR_H2B-BFP   608 ATATCAGCG TATCAGCG (1:1000)pLas + 20170 T4 E12 pL43 87.20% ATACAACTGCTT FALSE 2 ATTCCGA 3306pR_H2B-BFP   608 GCAACAGG (1:1000) pLas + 20170 T4 E12 sgRNA 88.00%ATACAACTGCTT FALSE 2 ATACAACTGCTT  176 pR_H2B-BFP   608 GCAACAGGGCAACAGG (1:1000) pLas + 20170 T4 F05 pL43 94.40% CCTGCAACGGG FALSE 2CGTCATA 2604 pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4 F05sgRNA 95.80% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 3619 pR_H2B-BFP   608ACTAGTTGG CTAGTTGG (1:1000) pLas + 20170 T4 F06 pL43 97.10% TCATATTACGAGFALSE 2 AGAGAGA 3573 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4F06 sgRNA 95.10% TCATATTACGAG FALSE 2 TCATATTACGAGT  252 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 F07 pL43 96.50%CCAGTACAAAC FALSE 2 AAGAGGA 3298 pR_H2B-BFP   608 CTACCTACG (1:1000)pLas + 20170 T4 F07 sgRNA 96.70% CCAGTACAAAC FALSE 2 CCAGTACAAACC  177pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000) pLas + 20170 T4 F08 pL4397.10% CCTGCAACGGG FALSE 2 CGTCATA 3554 pR_H2B-BFP   608 ACTAGTTGG(1:1000) pLas + 20170 T4 F08 sgRNA 95.90% CCTGCAACGGG FALSE 2CCTGCAACGGGA  279 pR_H2B-BFP   608 ACTAGTTGG CTAGTTGG (1:1000) pLas +20170 T4 F09 pL43 11.80% TCCACCGGCGA FALSE 8 CAATCGG  220 pR_H2B-BFP  608 AAGAGATCC (1:1000) pLas + 20170 T4 F09 pL43 13.20% AGAGCACTGCAFALSE 8 CCAGTTA  245 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4F09 pL43 16.50% TCATATTACGAG TRUE 8 AGAGAGA  306 pR_H2B-BFP   608TCAGTAGG (1:1000) pLas + 20170 T4 F09 pL43 18.00% CCAGTACAAAC FALSE 8AAGAGGA  334 pR_H2B-BFP   608 CTACCTACG (1:1000) pLas + 20170 T4 F09pL43 23.90% AGAGCACTGCA FALSE 8 AGGCTCT  443 pR_H2B-BFP   608 CTCCTTCA(1:1000) pLas + 20170 T4 F09 sgRNA 15.40% TCCACCGGCGA FALSE 8TCCACCGGCGAA   46 pR_H2B-BFP   608 AAGAGATCC AGAGATCC (1:1000) pLas +20170 T4 F09 sgRNA 21.10% AGAGCACTGCA FALSE 8 AGAGCACTGCAC   63pR_H2B-BFP   608 CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 F09 sgRNA60.70% CCAGTACAAAC FALSE 8 CCAGTACAAACC  181 pR_H2B-BFP   608 CTACCTACGTACCTACG (1:1000) pLas + 20170 T4 F11 pL43 93.80% CCTGCAACGGG FALSE 2CGTCATA 3904 pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4 F11sgRNA 95.40% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  922 pR_H2B-BFP   608ACTAGTTGG CTAGTTGG (1:1000) pLas + 20170 T4 F12 pL43 90.60% CCAGTACAAACFALSE 2 AAGAGGA 3835 pR_H2B-BFP   608 CTACCTACG (1:1000) pLas + 20170 T4F12 sgRNA 95.80% CCAGTACAAAC FALSE 2 CCAGTACAAACC  752 pR_H2B-BFP   608CTACCTACG TACCTACG (1:1000) pLas + 20170 T4 G05 pL43 96.50% CGCCGCCCCCGFALSE 2 CATGCGT 3920 pR_H2B-BFP   608 GACGCGACC (1:1000) pLas + 20170 T4G05 sgRNA 94.20% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  799 pR_H2B-BFP   608GACGCGACC ACGCGACC (1:1000) pLas + 20170 T4 G06 pL43 96.80% CGCCGCCCCCGFALSE 2 CATGCGT 4425 pR_H2B-BFP   608 GACGCGACC (1:1000) pLas + 20170 T4G06 sgRNA 95.30% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG 1567 pR_H2B-BFP   608GACGCGACC ACGCGACC (1:1000) pLas + 20170 T4 G07 pL43 95.20% AGAGCACTGCAFALSE 2 CCAGTTA 3330 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4G07 sgRNA 93.40% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 2697 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 G08 pL43 95.90% TCATATTACGAGFALSE 2 AGAGAGA 3450 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4G08 sgRNA 95.50% TCATATTACGAG FALSE 2 TCATATTACGAGT  191 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 G09 pL43 95.30%AGTAGTCCGGG FALSE 2 CCTCTTC 3053 pR_H2B-BFP   608 ATATCAGCG (1:1000)pLas + 20170 T4 G09 sgRNA 96.70% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  436pR_H2B-BFP   608 ATATCAGCG TATCAGCG (1:1000) pLas + 20170 T4 G10 pL4392.40% TCATATTACGAG FALSE 2 AGAGAGA 3573 pR_H2B-BFP   608 TCAGTAGG(1:1000) pLas + 20170 T4 G10 sgRNA 93.90% TCATATTACGAG FALSE 2TCATATTACGAGT  371 pR_H2B-BFP   608 TCAGTAGG CAGTAGG (1:1000) pLas +20170 T4 G11 pL43 94.00% CCAGTACAAAC FALSE 2 AAGAGGA 3619 pR_H2B-BFP  608 CTACCTACG (1:1000) pLas + 20170 T4 G11 sgRNA 96.00% CCAGTACAAACFALSE 2 CCAGTACAAACC  917 pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000)pLas + 20170 T4 G12 pL43 94.80% TCATATTACGAG FALSE 2 AGAGAGA 4109pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4 G12 sgRNA 95.60%TCATATTACGAG FALSE 2 TCATATTACGAGT  562 pR_H2B-BFP   608 TCAGTAGGCAGTAGG (1:1000) pLas + 20170 T4 H06 pL43 97.00% AGAGCACTGCA FALSE 2CCAGTTA 4164 pR_H2B-BFP   608 CTCCTTCA (1:1000) pLas + 20170 T4 H06sgRNA 94.00% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1025 pR_H2B-BFP   608CTCCTTCA TCCTTCA (1:1000) pLas + 20170 T4 H07 pL43 97.10% CCTGCAACGGGFALSE 2 CGTCATA 4657 pR_H2B-BFP   608 ACTAGTTGG (1:1000) pLas + 20170 T4H07 sgRNA 95.50% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1003 pR_H2B-BFP   608ACTAGTTGG CTAGTTGG (1:1000) pLas + 20170 T4 H08 pL43 97.80% TCATATTACGAGFALSE 2 AGAGAGA 4015 pR_H2B-BFP   608 TCAGTAGG (1:1000) pLas + 20170 T4H08 sgRNA 95.20% TCATATTACGAG FALSE 2 TCATATTACGAGT  460 pR_H2B-BFP  608 TCAGTAGG CAGTAGG (1:1000) pLas + 20170 T4 H09 pL43 96.00%AGTAGTCCGGG FALSE 2 CCTCTTC 3802 pR_H2B-BFP   608 ATATCAGCG (1:1000)pLas + 20170 T4 H09 sgRNA 95.10% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  818pR_H2B-BFP   608 ATATCAGCG TATCAGCG (1:1000) pLas + 20170 T4 H11 pL4395.90% CCAGTACAAAC FALSE 2 AAGAGGA 3996 pR_H2B-BFP   608 CTACCTACG(1:1000) pLas + 20170 T4 H11 sgRNA 95.10% CCAGTACAAAC FALSE 2CCAGTACAAACC  855 pR_H2B-BFP   608 CTACCTACG TACCTACG (1:1000) pLas +20170 T4 H12 pL43 74.40% CGCCGCCCCCG FALSE 2 CATGCGT 2767 pR_H2B-BFP  608 GACGCGACC (1:1000) pLas + 20170 T4 H12 sgRNA 94.40% CGCCGCCCCCGFALSE 2 CGCCGCCCCCGG  772 pR_H2B-BFP   608 GACGCGACC ACGCGACC (1:1000)pLas + pUC19 20170 T3 A09 pL43 84.80% AGTAGTCCGGG FALSE 2 CCTCTTC 2367(1:1000)   608 ATATCAGCG pLas + pUC19 20170 T3 A09 sgRNA 95.00%AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 2635 (1:1000)   608 ATATCAGCG TATCAGCGpLas + pUC19 20170 T3 A10 pL43 93.20% AGTAGTCCGGG TRUE 2 CCTCTTC 3551(1:1000)   608 ATATCAGCG pLas + pUC19 20170 T3 A10 sgRNA 95.30%CCAGTACAAAC TRUE 2 CCAGTACAAACC 1346 (1:1000)   608 CTACCTACG TACCTACGpLas + pUC19 20170 T3 A11 pL43 95.10% AGTAGTCCGGG FALSE 2 CCTCTTC 3567(1:1000)   608 ATATCAGCG pLas + pUC19 20170 T3 A11 sgRNA 95.10%AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 1483 (1:1000)   608 ATATCAGCG TATCAGCGpLas + pUC19 20170 T3 A12 pL43 96.50% AGAGCACTGCA FALSE 2 CCAGTTA 4125(1:1000)   608 CTCCTTCA pLas + pUC19 20170 T3 A12 sgRNA 95.30%AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1775 (1:1000)   608 CTCCTTCA TCCTTCApLas + pUC19 20170 T3 B09 pL43 74.90% TCATATTACGAG FALSE 2 AGAGAGA 2884(1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 B09 sgRNA 95.40%TCATATTACGAG FALSE 2 TCATATTACGAGT 1102 (1:1000)   608 TCAGTAGG CAGTAGGpLas + pUC19 20170 T3 B10 pL43 13.90% AGAGCACTGCA TRUE 4 CCAGTTA  353(1:1000)   608 CTCCTTCA pLas + pUC19 20170 T3 B10 pL43 20.20%TCATATTACGAG TRUE 4 AGAGAGA  512 (1:1000)   608 TCAGTAGG pLas + pUC1920170 T3 B10 pL43 30.80% CCAGTACAAAC TRUE 4 AAGAGGA  781 (1:1000)   608CTACCTACG pLas + pUC19 20170 T3 B10 sgRNA 89.30% CGCCGCCCCCG TRUE 4CGCCGCCCCCGG  134 (1:1000)   608 GACGCGACC ACGCGACC pLas + pUC19 20170T3 B11 pL43 92.00% TCATATTACGAG FALSE 2 AGAGAGA 4607 (1:1000)   608TCAGTAGG pLas + pUC19 20170 T3 B11 sgRNA 94.90% TCATATTACGAG FALSE 2TCATATTACGAGT  632 (1:1000)   608 TCAGTAGG CAGTAGG pLas + pUC19 20170 T3B12 pL43 10.30% TCATATTACGAG TRUE 4 AGAGAGA  343 (1:1000)   608 TCAGTAGGpLas + pUC19 20170 T3 B12 pL43 16.50% CCAGTACAAAC TRUE 4 AAGAGGA  549(1:1000)   608 CTACCTACG pLas + pUC19 20170 T3 B12 pL43 45.00%AGTAGTCCGGG FALSE 4 CCTCTTC 1500 (1:1000)   608 ATATCAGCG pLas + pUC1920170 T3 B12 sgRNA 95.80% AGTAGTCCGGG FALSE 4 AGTAGTCCGGGA 3532 (1:1000)  608 ATATCAGCG TATCAGCG pLas + pUC19 20170 T3 C09 pL43 93.70%TCATATTACGAG FALSE 2 AGAGAGA 3994 (1:1000)   608 TCAGTAGG pLas + pUC1920170 T3 C09 sgRNA 95.60% TCATATTACGAG FALSE 2 TCATATTACGAGT 1916(1:1000)   608 TCAGTAGG CAGTAGG pLas + pUC19 20170 T3 C10 pL43 15.70%AGAGCACTGCA TRUE 3 AGGCTCT  453 (1:1000)   608 CTCCTTCA pLas + pUC1920170 T3 C10 pL43 57.60% TCCACCGGCGA FALSE 3 CAATCGG 1658 (1:1000)   608AAGAGATCC pLas + pUC19 20170 T3 C10 sgRNA 94.60% TCCACCGGCGA FALSE 3TCCACCGGCGAA 1320 (1:1000)   608 AAGAGATCC AGAGATCC pLas + pUC19 20170T3 C11 pL43 71.90% AGAGCACTGCA FALSE 2 CCAGTTA 2500 (1:1000)   608CTCCTTCA pLas + pUC19 20170 T3 C11 sgRNA 95.30% AGAGCACTGCA FALSE 2AGAGCACTGCAC   61 (1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3C12 pL43 10.80% TCCACCGGCGA TRUE 5 CAATCGG  302 (1:1000)   608 AAGAGATCCpLas + pUC19 20170 T3 C12 pL43 16.20% TCATATTACGAG TRUE 5 AGAGAGA  456(1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 C12 pL43 17.10%CCAGTACAAAC TRUE 5 AAGAGGA  480 (1:1000)   608 CTACCTACG pLas + pUC1920170 T3 C12 pL43 40.00% AGAGCACTGCA FALSE 5 CCAGTTA 1123 (1:1000)   608CTCCTTCA pLas + pUC19 20170 T3 C12 sgRNA 94.90% AGAGCACTGCA FALSE 5AGAGCACTGCAC 1643 (1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3D09 pL43 11.80% CCAGTACAAAC TRUE 3 AAGAGGA  338 (1:1000)   608 CTACCTACGpLas + pUC19 20170 T3 D09 pL43 57.60% TCATATTACGAG FALSE 3 AGAGAGA 1647(1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 D09 sgRNA 91.00%TCATATTACGAG FALSE 3 TCATATTACGAGT  783 (1:1000)   608 TCAGTAGG CAGTAGGpLas + pUC19 20170 T3 D10 pL43 94.70% CGCCGCCCCCG FALSE 2 CATGCGT 4961(1:1000)   608 GACGCGACC pLas + pUC19 20170 T3 D10 sgRNA 95.70%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  991 (1:1000)   608 GACGCGACC ACGCGACCpLas + pUC19 20170 T3 D11 pL43 94.80% AGTAGTCCGGG FALSE 2 CCTCTTC 4155(1:1000)   608 ATATCAGCG pLas + pUC19 20170 T3 D11 sgRNA 95.70%AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  933 (1:1000)   608 ATATCAGCG TATCAGCGpLas + pUC19 20170 T3 D12 pL43 76.80% TCATATTACGAG TRUE 2 AGAGAGA 2695(1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 D12 sgRNA 94.90%CCAGTACAAAC TRUE 2 CCAGTACAAACC 1313 (1:1000)   608 CTACCTACG TACCTACGpLas + pUC19 20170 T3 E09 pL43 91.10% TCATATTACGAG FALSE 2 AGAGAGA 3176(1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 E09 sgRNA 96.10%TCATATTACGAG FALSE 2 TCATATTACGAGT  928 (1:1000)   608 TCAGTAGG CAGTAGGpLas + pUC19 20170 T3 E10 pL43 10.50% AGAGCACTGCA TRUE 7 AGGCTCT  288(1:1000)   608 CTCCTTCA pLas + pUC19 20170 T3 E10 pL43 12.50%AGAGCACTGCA TRUE 7 CCAGTTA  343 (1:1000)   608 CTCCTTCA pLas + pUC1920170 T3 E10 pL43 13.60% TCCACCGGCGA TRUE 7 ACTGGCT  371 (1:1000)   608AAGAGATCC pLas + pUC19 20170 T3 E10 pL43 13.70% TCATATTACGAG TRUE 7AGAGAGA  375 (1:1000)   608 TCAGTAGG pLas + pUC19 20170 T3 E10 pL4313.80% TCCACCGGCGA TRUE 7 CAATCGG  376 (1:1000)   608 AAGAGATCC pLas +pUC19 20170 T3 E10 pL43 19.30% CCAGTACAAAC TRUE 7 AAGAGGA  529 (1:1000)  608 CTACCTACG pLas + pUC19 20170 T3 E10 sgRNA 85.30% CCTGCAACGGG TRUE7 CCTGCAACGGGA  320 (1:1000)   608 ACTAGTTGG CTAGTTGG pLas + pUC19 20170T3 E11 pL43 94.90% CCAGTACAAAC FALSE 2 AAGAGGA 3540 (1:1000)   608CTACCTACG pLas + pUC19 20170 T3 E11 sgRNA 95.40% CCAGTACAAAC FALSE 2CCAGTACAAACC  722 (1:1000)   608 CTACCTACG TACCTACG pLas + pUC19 20170T3 E12 pL43 93.00% CGCCGCCCCCG FALSE 2 CATGCGT 3594 (1:1000)   608GACGCGACC pLas + pUC19 20170 T3 E12 sgRNA 93.80% CGCCGCCCCCG FALSE 2CGCCGCCCCCGG   61 (1:1000)   608 GACGCGACC ACGCGACC pLas + pUC19 20170T3 F09 pL43 95.60% CGCCGCCCCCG FALSE 2 CATGCGT 3644 (1:1000)   608GACGCGACC pLas + pUC19 20170 T3 F09 sgRNA 94.50% CGCCGCCCCCG FALSE 2CGCCGCCCCCGG  242 (1:1000)   608 GACGCGACC ACGCGACC pLas + pUC19 20170T3 F10 pL43 95.40% CCTGCAACGGG FALSE 2 CGTCATA 4252 (1:1000)   608ACTAGTTGG pLas + pUC19 20170 T3 F10 sgRNA 94.30% CCTGCAACGGG FALSE 2CCTGCAACGGGA  644 (1:1000)   608 ACTAGTTGG CTAGTTGG pLas + pUC19 20170T3 F11 pL43 12.20% AGAGCACTGCA TRUE 3 AGGCTCT  360 (1:1000)   608CTCCTTCA pLas + pUC19 20170 T3 F11 pL43 72.70% CCTGCAACGGG FALSE 3CGTCATA 2141 (1:1000)   608 ACTAGTTGG pLas + pUC19 20170 T3 F11 sgRNA94.50% CCTGCAACGGG FALSE 3 CTGCAACGGGA 2120 (1:1000)   608 ACTAGTTGGCTAGTTGG pLas + pUC19 20170 T3 F12 pL43 16.30% AGAGCACTGCA TRUE 3AGGCTCT  460 (1:1000)   608 CTCCTTCA pLas + pUC19 20170 T3 F12 pL4367.60% CCTGCAACGGG FALSE 3 CGTCATA 1906 (1:1000)   608 ACTAGTTGG pLas +pUC19 20170 T3 F12 sgRNA 95.50% CCTGCAACGGG FALSE 3 CCTGCAACGGGA  708(1:1000)   608 ACTAGTTGG CTAGTTGG pLas + pUC19 20170 T3 G09 pL43 13.20%AGAGCACTGCA FALSE 6 CCAGTTA  331 (1:1000)   608 CTCCTTCA pLas + pUC1920170 T3 G09 pL43 16.10% CCAGTACAAAC TRUE 6 AAGAGGA  404 (1:1000)   608CTACCTACG pLas + pUC19 20170 T3 G09 pL43 21.70% TCCACCGGCGA FALSE 6CAATCGG  546 (1:1000)   608 AAGAGATCC pLas + pUC19 20170 T3 G09 pL4324.30% TCATATTACGAG TRUE 6 AGAGAGA  612 (1:1000)   608 TCAGTAGG pLas +pUC19 20170 T3 G09 sgRNA 34.80% AGAGCACTGCA FALSE 6 AGAGCACTGCAC   32(1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3 G09 sgRNA 58.70%TCCACCGGCGA FALSE 6 TCCACCGGCGAA   54 (1:1000)   608 AAGAGATCC AGAGATCCpLas + pUC19 20170 T3 G10 pL43 91.70% ATACAACTGCTT FALSE 2 ATTCCGA 4180(1:1000)   608 GCAACAGG pLas + pUC19 20170 T3 G10 sgRNA 93.80%ATACAACTGCTT FALSE 2 ATACAACTGCTT 1091 (1:1000)   608 GCAACAGG GCAACAGGpLas + pUC19 20170 T3 G11 pL43 18.10% TCCACCGGCGA TRUE 4 CAATCGG  434(1:1000)   608 AAGAGATCC pLas + pUC19 20170 T3 G11 pL43 23.10%CCAGTACAAAC TRUE 4 AAGAGGA  553 (1:1000)   608 CTACCTACG pLas + pUC1920170 T3 G11 pL43 29.40% AGAGCACTGCA FALSE 4 CCAGTTA  703 (1:1000)   608CTCCTTCA pLas + pUC19 20170 T3 G11 sgRNA 93.70% AGAGCACTGCA FALSE 4AGAGCACTGCAC 6346 (1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3G12 pL43 92.20% AGAGCACTGCA FALSE 2 CCAGTTA 4422 (1:1000)   608 CTCCTTCApLas + pUC19 20170 T3 G12 sgRNA 95.40% AGAGCACTGCA FALSE 2 AGAGCACTGCAC1195 (1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3 H09 pL4395.70% AGAGCACTGCA FALSE 2 CCAGTTA 4388 (1:1000)   608 CTCCTTCA pLas +pUC19 20170 T3 H09 sgRNA 95.90% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  845(1:1000)   608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T3 H10 pL43 94.30%AGTAGTCCGGG FALSE 2 CCTCTTC 3658 (1:1000)   608 ATATCAGCG pLas + pUC1920170 T3 H10 sgRNA 96.40% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 1427 (1:1000)  608 ATATCAGCG TATCAGCG pLas + pUC19 20170 T3 H11 pL43 95.10%TCATATTACGAG FALSE 2 AGAGAGA 4519 (1:1000)   608 TCAGTAGG pLas + pUC1920170 T3 H11 sgRNA 95.80% TCATATTACGAG FALSE 2 TCATATTACGAGT  364(1:1000)   608 TCAGTAGG CAGTAGG pLas + pUC19 20170 T3 H12 pL43 93.60%CGCCGCCCCCG FALSE 2 CATGCGT 4552 (1:1000)   608 GACGCGACC pLas + pUC1920170 T3 H12 sgRNA 94.40% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  167 (1:1000)  608 GACGCGACC ACGCGACC pLas + pUC19 20170 T4 A01 pL43 96.20%AGAGCACTGCA FALSE 2 CCAGTTA 4068 (1:1000)   608 CTCCTTCA pLas + pUC1920170 T4 A01 sgRNA 95.00% AGAGCACTGCA FALSE 2 AGAGCACTGCAC 1106 (1:1000)  608 CTCCTTCA TCCTTCA pLas + pUC19 20170 T4 A02 pL43 94.70% AGTAGTCCGGGFALSE 2 CCTCTTC 3081 (1:1000)   608 ATATCAGCG pLas + pUC19 20170 T4 A02sgRNA 94.80% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA  311 (1:1000)   608ATATCAGCG TATCAGCG pLas + pUC19 20170 T4 A03 pL43 91.20% CCAGTACAAACFALSE 2 AAGAGGA 2564 (1:1000)   608 CTACCTACG pLas + pUC19 20170 T4 A03sgRNA 96.10% CCAGTACAAAC FALSE 2 CCAGTACAAACC  123 (1:1000)   608CTACCTACG TACCTACG pLas + pUC19 20170 T4 A04 pL43 94.10% ATACAACTGCTTFALSE 2 ATTCCGA 3348 (1:1000)   608 GCAACAGG pLas + pUC19 20170 T4 A04sgRNA 94.70% ATACAACTGCTT FALSE 2 ATACAACTGCTT 1009 (1:1000)   608GCAACAGG GCAACAGG pLas + pUC19 20170 T4 B01 pL43 96.40% CGCCGCCCCCGFALSE 2 CATGCGT 4085 (1:1000)   608 GACGCGACC pLas + pUC19 20170 T4 B01sgRNA 95.30% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  946 (1:1000)   608GACGCGACC ACGCGACC pLas + pUC19 20170 T4 B02 pL43 93.10% CGCCGCCCCCGFALSE 2 CATGCGT 3533 (1:1000)   608 GACGCGACC pLas + pUC19 20170 T4 B02sgRNA 95.90% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG 1965 (1:1000)   608GACGCGACC ACGCGACC pLas + pUC19 20170 T4 B03 pL43 93.90% AGTAGTCCGGGFALSE 2 CCTCTTC 3112 (1:1000)   608 ATATCAGCG pLas + pUC19 20170 T4 B03sgRNA 95.60% AGTAGTCCGGG FALSE 2 AGTAGTCCGGGA 1585 (1:1000)   608ATATCAGCG TATCAGCG pLas + pUC19 20170 T4 B04 pL43 95.10% CGCCGCCCCCGFALSE 2 CATGCGT 3625 (1:1000)   608 GACGCGACC pLas + pUC19 20170 T4 B04sgRNA 96.10% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  374 (1:1000)   608GACGCGACC ACGCGACC pLas + pUC19 20170 T4 C01 pL43 95.10% AGAGCACTGCAFALSE 2 CCAGTTA 3670 (1:1000)   608 CTCCTTCA pLas + pUC19 20170 T4 C01sgRNA 93.70% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  848 (1:1000)   608CTCCTTCA TCCTTCA pLas + pUC19 20170 T4 C02 pL43 85.10% CGCCGCCCCCG FALSE2 CATGCGT 2940 (1:1000)   608 GACGCGACC pLas + pUC19 20170 T4 C02 sgRNA94.80% CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  454 (1:1000)   608 GACGCGACCACGCGACC pLas + pUC19 20170 T4 C03 pL43 90.50% CCTGCAACGGG FALSE 2CGTCATA 3055 (1:1000)   608 ACTAGTTGG pLas + pUC19 20170 T4 C03 sgRNA95.50% CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1330 (1:1000)   608 ACTAGTTGGCTAGTTGG pLas + pUC19 20170 T4 C04 pL43 92.00% AGAGCACTGCA FALSE 2CCAGTTA 2854 (1:1000)   608 CTCCTTCA pLas + pUC19 20170 T4 C04 sgRNA93.70% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  593 (1:1000)   608 CTCCTTCATCCTTCA pLas + pUC19 20170 T4 D01 pL43 96.50% CCTGCAACGGG FALSE 2CGTCATA 3679 (1:1000)   608 ACTAGTTGG pLas + pUC19 20170 T4 D01 sgRNA95.80% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  692 (1:1000)   608 ACTAGTTGGCTAGTTGG pLas, 11h 20170 T2 B05 pL43 96.80% CCTGCAACGGG TRUE 2 CGTCATA3770 packaging   608 ACTAGTTGG pLas, 11h 20170 T2 B05 sgRNA 95.30%AGAGCACTGCA TRUE 2 AGAGCACTGCAC 1369 packaging   608 CTCCTTCA TCCTTCApLas, 11h 20170 T2 B06 pL43 83.60% CCTGCAACGGG FALSE 2 CGTCATA 2362packaging   608 ACTAGTTGG pLas, 11h 20170 T2 B06 sgRNA 95.70%CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1597 packaging   608 ACTAGTTGG CTAGTTGGpLas, 11h 20170 T2 B07 pL43 96.30% TCATATTACGAG TRUE 2 AGAGAGA 3817packaging   608 TCAGTAGG pLas, 11h 20170 T2 B07 sgRNA 94.40%ATACAACTGCTT TRUE 2 ATACAACTGCTT 1232 packaging   608 GCAACAGG GCAACAGGpLas, 11h 20170 T2 B08 pL43 96.90% CGCCGCCCCCG FALSE 2 CATGCGT 4509packaging   608 GACGCGACC pLas, 11h 20170 T2 B08 sgRNA 95.90%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  649 packaging   608 GACGCGACC ACGCGACCpLas, 11h 20170 T2 B09 pL43 87.80% CGCCGCCCCCG FALSE 2 CATGCGT 3644packaging   608 GACGCGACC pLas, 11h 20170 T2 B09 sgRNA 96.10%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  416 packaging   608 GACGCGACC ACGCGACCpLas, 11h 20170 T2 B10 pL43 46.40% CCAGTACAAAC FALSE 4 AAGAGGA 1539packaging   608 CTACCTACG pLas, 11h 20170 T2 B10 pL43 47.30%TCATATTACGAG FALSE 4 AGAGAGA 1570 packaging   608 TCAGTAGG pLas, 11h20170 T2 B10 sgRNA 40.90% TCATATTACGAG FALSE 4 TCATATTACGAGT   74packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2 B10 sgRNA 51.90%CCAGTACAAAC FALSE 4 CCAGTACAAACC   94 packaging   608 CTACCTACG TACCTACGpLas, 11h 20170 T2 B11 pL43 75.30% CGCCGCCCCCG FALSE 2 CATGCGT 3109packaging   608 GACGCGACC pLas, 11h 20170 T2 B11 sgRNA 95.80%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  684 packaging   608 GACGCGACC ACGCGACCpLas, 11h 20170 T2 B12 pL43 69.70% CCTGCAACGGG FALSE 2 CGTCATA 2542packaging   608 ACTAGTTGG pLas, 11h 20170 T2 B12 sgRNA 95.00%CCTGCAACGGG FALSE 2 CCTGCAACGGGA 1034 packaging   608 ACTAGTTGG CTAGTTGGpLas, 11h 20170 T2 C05 pL43 97.50% TCCACCGGCGA TRUE 2 CAATCGG 3989packaging   608 AAGAGATCC pLas, 11h 20170 T2 C05 sgRNA 94.70%AGTAGTCCGGG TRUE 2 AGTAGTCCGGGA 1079 packaging   608 ATATCAGCG TATCAGCGpLas, 11h 20170 T2 C06 pL43 95.80% ATACAACTGCTT TRUE 2 ATTCCGA 4057packaging   608 GCAACAGG pLas, 11h 20170 T2 C06 sgRNA 94.40% AGTAGTCCGGGTRUE 2 AGTAGTCCGGGA  725 packaging   608 ATATCAGCG TATCAGCG pLas, 11h20170 T2 C07 pL43 94.70% AGTAGTCCGGG TRUE 2 CCTCTTC 3449 packaging   608ATATCAGCG pLas, 11h 20170 T2 C07 sgRNA 94.00% ATACAACTGCTT TRUE 2ATACAACTGCTT  676 packaging   608 GCAACAGG GCAACAGG pLas, 11h 20170 T2C08 pL43 96.50% TCCACCGGCGA TRUE 2 CAATCGG 4264 packaging   608AAGAGATCC pLas, 11h 20170 T2 C08 sgRNA 95.70% CCTGCAACGGG TRUE 2CCTGCAACGGGA  737 packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2C09 pL43 96.00% TCCACCGGCGA FALSE 2 CAATCGG 4160 packaging   608AAGAGATCC pLas, 11h 20170 T2 C09 sgRNA 93.90% TCCACCGGCGA FALSE 2TCCACCGGCGAA  726 packaging   608 AAGAGATCC AGAGATCC pLas, 11h 20170 T2C10 pL43 12.20% AGTAGTCCGGG TRUE 6 CCTCTTC  426 packaging   608ATATCAGCG pLas, 11h 20170 T2 C10 pL43 33.00% AGAGCACTGCA FALSE 6 AGGCTCT1152 packaging   608 CTCCTTCA pLas, 11h 20170 T2 C10 pL43 46.60%TCCACCGGCGA FALSE 6 CAATCGG 1625 packaging   608 AAGAGATCC pLas, 11h20170 T2 C10 sgRNA 22.00% CGCCGCCCCCG TRUE 6 CGCCGCCCCCGG  172 packaging  608 GACGCGACC ACGCGACC pLas, 11h 20170 T2 C10 sgRNA 31.50% AGAGCACTGCAFALSE 6 AGAGCACTGCAC  246 packaging   608 CTCCTTCA TCCTTCA pLas, 11h20170 T2 C10 sgRNA 38.40% TCCACCGGCGA FALSE 6 TCCACCGGCGAA  300packaging   608 AAGAGATCC AGAGATCC pLas, 11h 20170 T2 C11 pL43 43.80%AGTAGTCCGGG TRUE 4 CCTCTTC 1695 packaging   608 ATATCAGCG pLas, 11h20170 T2 C11 pL43 52.20% ATACAACTGCTT FALSE 4 ATTCCGA 2019 packaging  608 GCAACAGG pLas, 11h 20170 T2 C11 sgRNA 32.20% ATACAACTGCTT FALSE 4ATACAACTGCTT  284 packaging   608 GCAACAGG GCAACAGG pLas, 11h 20170 T2C11 sgRNA 63.80% CGCCGCCCCCG TRUE 4 CGCCGCCCCCGG  563 packaging   608GACGCGACC ACGCGACC pLas, 11h 20170 T2 C12 pL43 11.10% TCCACCGGCGA TRUE 4CAATCGG  322 packaging   608 AAGAGATCC pLas, 11h 20170 T2 C12 pL4317.90% CCAGTACAAAC TRUE 4 AAGAGGA  518 packaging   608 CTACCTACGpLas, 11h 20170 T2 C12 pL43 38.60% ATACAACTGCTT FALSE 4 ATTCCGA 1115packaging   608 GCAACAGG pLas, 11h 20170 T2 C12 sgRNA 93.70%ATACAACTGCTT FALSE 4 ATACAACTGCTT  417 packaging   608 GCAACAGG GCAACAGGpLas, 11h 20170 T2 D05 pL43 97.20% AGAGCACTGCA TRUE 2 CCAGTTA 4097packaging   608 CTCCTTCA pLas, 11h 20170 T2 D05 sgRNA 94.30%ATACAACTGCTT TRUE 2 ATACAACTGCTT  834 packaging   608 GCAACAGG GCAACAGGpLas, 11h 20170 T2 D07 pL43 97.60% TCATATTACGAG FALSE 2 AGAGAGA 4546packaging   608 TCAGTAGG pLas, 11h 20170 T2 D07 sgRNA 93.20%TCATATTACGAG FALSE 2 TCATATTACGAGT  205 packaging   608 TCAGTAGG CAGTAGGpLas, 11h 20170 T2 D08 pL43 92.00% AGAGCACTGCA TRUE 2 CCAGTTA 2574packaging   608 CTCCTTCA pLas, 11h 20170 T2 D08 sgRNA 93.80% CCAGTACAAACTRUE 2 CCAGTACAAACC  456 packaging   608 CTACCTACG TACCTACG pLas, 11h20170 T2 D09 pL43 96.80% TCATATTACGAG FALSE 2 AGAGAGA 4523 packaging  608 TCAGTAGG pLas, 11h 20170 T2 D09 sgRNA 94.70% TCATATTACGAG FALSE 2TCATATTACGAGT  304 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2D10 pL43 95.60% CGCCGCCCCCG TRUE 2 CATGCGT 4801 packaging   608GACGCGACC pLas, 11h 20170 T2 D10 sgRNA 92.70% ATACAACTGCTT TRUE 2ATACAACTGCTT  190 packaging   608 GCAACAGG GCAACAGG pLas, 11h 20170 T2D11 pL43 85.10% CCAGTACAAAC FALSE 2 AAGAGGA 3220 packaging   608CTACCTACG pLas, 11h 20170 T2 D11 sgRNA 95.50% CCAGTACAAAC FALSE 2CCAGTACAAACC  778 packaging   608 CTACCTACG TACCTACG pLas, 11h 20170 T2D12 pL43 94.90% ATACAACTGCTT FALSE 2 ATTCCGA 4344 packaging   608GCAACAGG pLas, 11h 20170 T2 D12 sgRNA 96.50% ATACAACTGCTT FALSE 2ATACAACTGCTT  495 packaging   608 GCAACAGG GCAACAGG pLas, 11h 20170 T2E06 pL43 97.40% TCATATTACGAG FALSE 2 AGAGAGA 4683 packaging   608TCAGTAGG pLas, 11h 20170 T2 E06 sgRNA 96.70% TCATATTACGAG FALSE 2TCATATTACGAGT  468 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2E07 pL43 95.50% TCATATTACGAG FALSE 2 AGAGAGA 3586 packaging   608TCAGTAGG pLas, 11h 20170 T2 E07 sgRNA 93.60% TCATATTACGAG FALSE 2TCATATTACGAGT  775 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2E09 pL43 95.60% ATACAACTGCTT TRUE 2 ATTCCGA 4076 packaging   608GCAACAGG pLas, 11h 20170 T2 E09 sgRNA 96.30% AGTAGTCCGGG TRUE 2AGTAGTCCGGGA  441 packaging   608 ATATCAGCG TATCAGCG pLas, 11h 20170 T2E10 pL43 94.00% CGCCGCCCCCG FALSE 2 CATGCGT 4581 packaging   608GACGCGACC pLas, 11h 20170 T2 E10 sgRNA 94.90% CGCCGCCCCCG FALSE 2CGCCGCCCCCGG  542 packaging   608 GACGCGACC ACGCGACC pLas, 11h 20170 T2E11 pL43 63.80% AGAGCACTGCA FALSE 2 CCAGTTA 1983 packaging   608CTCCTTCA pLas, 11h 20170 T2 E11 sgRNA 94.60% AGAGCACTGCA FALSE 2AGAGCACTGCAC  913 packaging   608 CTCCTTCA TCCTTCA pLas, 11h 20170 T2E12 pL43 91.40% CGCCGCCCCCG TRUE 2 CATGCGT 3833 packaging   608GACGCGACC pLas, 11h 20170 T2 E12 sgRNA 96.00% TCATATTACGAG TRUE 2TCATATTACGAGT  868 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2F05 pL43 97.00% CCTGCAACGGG FALSE 2 CGTCATA 4286 packaging   608ACTAGTTGG pLas, 11h 20170 T2 F05 sgRNA 96.30% CCTGCAACGGG FALSE 2CCTGCAACGGGA  209 packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2F06 pL43 96.60% CGCCGCCCCCG TRUE 2 CATGCGT 3706 packaging   608GACGCGACC pLas, 11h 20170 T2 F06 sgRNA 95.70% CCTGCAACGGG TRUE 2CCTGCAACGGGA  309 packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2F07 pL43 96.80% AGAGCACTGCA TRUE 2 CCAGTTA 4093 packaging   608 CTCCTTCApLas, 11h 20170 T2 F07 sgRNA 94.50% AGTAGTCCGGG TRUE 2 AGTAGTCCGGGA  361packaging   608 ATATCAGCG TATCAGCG pLas, 11h 20170 T2 F08 pL43 97.40%CCTGCAACGGG FALSE 2 CGTCATA 3440 packaging   608 ACTAGTTGG pLas, 11h20170 T2 F08 sgRNA 94.20% CCTGCAACGGG FALSE 2 CCTGCAACGGGA   98packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2 F10 pL43 94.60%CCTGCAACGGG FALSE 2 CGTCATA 4602 packaging   608 ACTAGTTGG pLas, 11h20170 T2 F10 sgRNA 95.40% CCTGCAACGGG FALSE 2 CCTGCAACGGGA  518packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2 F11 pL43 15.50%AGAGCACTGCA FALSE 4 AGGCTCT  626 packaging   608 CTCCTTCA pLas, 11h20170 T2 F11 pL43 80.40% TCCACCGGCGA TRUE 4 CAATCGG 3245 packaging   608AAGAGATCC pLas, 11h 20170 T2 F11 sgRNA 10.20% AGAGCACTGCA FALSE 4AGAGCACTGCAC   79 packaging   608 CTCCTTCA TCCTTCA pLas, 11h 20170 T2F11 sgRNA 85.50% TCATATTACGAG TRUE 4 TCATATTACGAGT  664 packaging   608TCAGTAGG CAGTAGG pLas, 11h 20170 T2 F12 pL43 95.60% AGAGCACTGCA FALSE 2CCAGTTA 4146 packaging   608 CTCCTTCA pLas, 11h 20170 T2 F12 sgRNA95.30% AGAGCACTGCA FALSE 2 AGAGCACTGCAC  486 packaging   608 CTCCTTCATCCTTCA pLas, 11h 20170 T2 G05 pL43 97.20% CGCCGCCCCCG FALSE 2 CATGCGT3720 packaging   608 GACGCGACC pLas, 11h 20170 T2 G05 sgRNA 95.10%CGCCGCCCCCG FALSE 2 CGCCGCCCCCGG  509 packaging   608 GACGCGACC ACGCGACCpLas, 11h 20170 T2 G06 pL43 97.30% CCTGCAACGGG TRUE 2 CGTCATA 3869packaging   608 ACTAGTTGG pLas, 11h 20170 T2 G06 sgRNA 95.80%ATACAACTGCTT TRUE 2 ATACAACTGCTT  159 packaging   608 GCAACAGG GCAACAGGpLas, 11h 20170 T2 G07 pL43 85.90% CCTGCAACGGG FALSE 2 CGTCATA 1831packaging   608 ACTAGTTGG pLas, 11h 20170 T2 G07 sgRNA 95.00%CCTGCAACGGG FALSE 2 CCTGCAACGGGA 4525 packaging   608 ACTAGTTGG CTAGTTGGpLas, 11h 20170 T2 G08 pL43 95.30% CCAGTACAAAC FALSE 2 AAGAGGA 3386packaging   608 CTACCTACG pLas, 11h 20170 T2 G08 sgRNA 94.70%CCAGTACAAAC FALSE 2 CCAGTACAAACC  160 packaging   608 CTACCTACG TACCTACGpLas, 11h 20170 T2 G09 pL43 95.20% CCTGCAACGGG FALSE 2 CGTCATA 4430packaging   608 ACTAGTTGG pLas, 11h 20170 T2 G09 sgRNA 96.00%CCTGCAACGGG FALSE 2 CCTGCAACGGGA  427 packaging   608 ACTAGTTGG CTAGTTGGpLas, 11h 20170 T2 G10 pL43 92.50% AGAGCACTGCA TRUE 2 CCAGTTA 4090packaging   608 CTCCTTCA pLas, 11h 20170 T2 G10 sgRNA 95.80% AGTAGTCCGGGTRUE 2 AGTAGTCCGGGA  915 packaging   608 ATATCAGCG TATCAGCG pLas, 11h20170 T2 G11 pL43 95.90% TCATATTACGAG FALSE 2 AGAGAGA 3667 packaging  608 TCAGTAGG pLas, 11h 20170 T2 G11 sgRNA 94.80% TCATATTACGAG FALSE 2TCATATTACGAGT  307 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2G12 pL43 92.40% AGAGCACTGCA FALSE 2 CCAGTTA 4131 packaging   608CTCCTTCA pLas, 11h 20170 T2 G12 sgRNA 95.70% AGAGCACTGCA FALSE 2AGAGCACTGCAC  132 packaging   608 CTCCTTCA TCCTTCA pLas, 11h 20170 T2H05 pL43 96.00% AGTAGTCCGGG TRUE 2 CCTCTTC 3216 packaging   608ATATCAGCG pLas, 11h 20170 T2 H05 sgRNA 95.40% CCTGCAACGGG TRUE 2CCTGCAACGGGA 1403 packaging   608 ACTAGTTGG CTAGTTGG pLas, 11h 20170 T2H06 pL43 97.10% TCATATTACGAG FALSE 2 AGAGAGA 4069 packaging   608TCAGTAGG pLas, 11h 20170 T2 H06 sgRNA 95.00% TCATATTACGAG FALSE 2TCATATTACGAGT  892 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2H07 pL43 97.10% AGAGCACTGCA FALSE 2 CCAGTTA 4126 packaging   608CTCCTTCA pLas, 11h 20170 T2 H07 sgRNA 94.50% AGAGCACTGCA FALSE 2AGAGCACTGCAC 1827 packaging   608 CTCCTTCA TCCTTCA pLas, 11h 20170 T2H08 pL43 97.00% TCATATTACGAG FALSE 2 AGAGAGA 4237 packaging   608TCAGTAGG pLas, 11h 20170 T2 H08 sgRNA 95.80% TCATATTACGAG FALSE 2TCATATTACGAGT 1067 packaging   608 TCAGTAGG CAGTAGG pLas, 11h 20170 T2H09 pL43 19.00% TCATATTACGAG TRUE 3 AGAGAGA  337 packaging   608TCAGTAGG pLas, 11h 20170 T2 H09 pL43 35.90% CCAGTACAAAC TRUE 3 AAGAGGA 636 packaging   608 CTACCTACG pLas, 11h 20170 T2 H09 sgRNA 94.10%AGAGCACTGCA TRUE 3 AGAGCACTGCAC 9185 packaging   608 CTCCTTCA TCCTTCApLas, 11h 20170 T2 H11 pL43 17.30% AGAGCACTGCA TRUE 5 CCAGTTA  426packaging   608 CTCCTTCA pLas, 11h 20170 T2 H11 pL43 18.50% TCATATTACGAGTRUE 5 AGAGAGA  456 packaging   608 TCAGTAGG pLas, 11h 20170 T2 H11 pL4320.30% TCCACCGGCGA TRUE 5 CAATCGG  502 packaging   608 AAGAGATCCpLas, 11h 20170 T2 H11 pL43 21.10% CCAGTACAAAC TRUE 5 AAGAGGA  522packaging   608 CTACCTACG pLas, 11h 20170 T2 H11 sgRNA 95.40%CGCCGCCCCCG TRUE 5 CGCCGCCCCCGG 3088 packaging   608 GACGCGACC ACGCGACCpLas, 11h 20170 T2 H12 pL43 90.50% AGAGCACTGCA FALSE 2 CCAGTTA 3677packaging   608 CTCCTTCA pLas, 11h 20170 T2 H12 sgRNA 95.20% AGAGCACTGCAFALSE 2 AGAGCACTGCAC  598 packaging   608 CTCCTTCA TCCTTCA pLas + pR_LG20170 T1 A01 pL42_ 97.00% CAAGGAGGACG FALSE 2 CTATATATGACC 4947 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 A01 sgRNA 89.50% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 3492 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 A02 pL42_ 97.20% GCCGTGCCGTA FALSE 2 GACATGCGTAGC 5560 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 A02 sgRNA 89.80% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 3437 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 A03 pL42_ 96.20% TGTACTCCAGCT FALSE 2 AGCTCTGACACA 5246 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 A03 sgRNA 89.70% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4470 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 A04 pL42_ 96.30% AAGGAGGACGG FALSE 2 TCCAAAGAGACA 5671 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 A04 sgRNA 90.00% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 4800 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 A05 pL42_ 97.20% CAAGGAGGACG FALSE 2 CTATATATGACC 5430 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 A05 sgRNA 91.50% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 5875 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 A06 pL42_ 96.90% GCCGTGCCGTA FALSE 2 AGATGATAACGG 5589 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 A06 sgRNA 89.20% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 5212 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 A07 pL42_ 96.50% AAGGAGGACGG FALSE 2 TCCAAAGAGACA 5717 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 A07 sgRNA 89.70% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 5701 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 A08 pL42_ 96.40% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5163 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 A08 sgRNA 86.70% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4141 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 A09 pL42_ 96.40% GTACAGCTAAG FALSE 2 AGCACGGAGACA 5313 (1:10)  924 pool1 TTAAACTCG pLas + pR_LG 20170 T1 A09 sgRNA 88.80% GTACAGCTAAGFALSE 2 GTACAGCTAAGT 3082 (1:10)   924 TTAAACTCG TAAACTCG pLas + pR_LG20170 T1 A10 pL42_ 96.80% AAGGAGGACGG FALSE 2 GAATCCGCTCGC 5743 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 A10 sgRNA 90.60% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 4341 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 A11 pL42_ 96.30% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5168 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 A11 sgRNA 88.20% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4120 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 B01 pL42_ 97.10% GTCCGTTCGACA FALSE 2 CTCAATTTACAG 5092 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 B01 sgRNA 87.20% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 2546 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 B02 pL42_ 96.90% AAGGAGGACGG FALSE 2 CTAGTGTCCACA 5442 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 B02 sgRNA 91.80% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 3877 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 B03 pL42_ 97.00% GCCGTGCCGTA FALSE 2 GACAACGAGAAC 5605 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 B03 sgRNA 90.70% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 4741 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 B04 pL42_ 96.90% GGACGCTAAAC FALSE 2 AGAGACTTCACA 5232 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 B04 sgRNA 90.00% GGACGCTAAACFALSE 2 GGACGCTAAACC 4847 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 B05 pL42_ 96.90% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5302 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 B05 sgRNA 88.00% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 3966 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 B06 pL42_ 97.40% GGACGCTAAAC FALSE 2 GACAGGCTACCT 5500 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 B06 sgRNA 91.60% GGACGCTAAACFALSE 2 GGACGCTAAACC 4698 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 B07 pL42_ 96.30% AAGGAGGACGG FALSE 2 GAATGAACCACG 6121 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 B07 sgRNA 91.90% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 5193 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 B08 pL42_ 97.40% CAACATCCTGG FALSE 2 GAACGCGAAAGC 5179 (1:10)  924 pool1 GGCACAAGC pLas + pR_LG 20170 T1 B08 sgRNA 90.70% CAACATCCTGGFALSE 2 CAACATCCTGGG 4939 (1:10)   924 GGCACAAGC GCACAAGC pLas + pR_LG20170 T1 B09 pL42_ 97.00% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5307 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 B09 sgRNA 88.30% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4370 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 B10 pL42_ 95.90% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5921 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 B10 sgRNA 90.20% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 4136 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 B11 pL42_ 97.20% GCCGTGCCGTA FALSE 2 GACATGCGTAGC 5359 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 B11 sgRNA 89.30% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 3745 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 C01 pL42_ 97.00% AAGGAGGACGG FALSE 2 TCCAAAGAGACA 5345 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 C01 sgRNA 89.30% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 5246 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 C02 pL42_ 97.80% GCTGCTTGCGAT FALSE 2 AGCAGCCCTAGC 5419 (1:10)  924 pool1 ACCAATAG pLas + pR_LG 20170 T1 C02 sgRNA 89.90% GCTGCTTGCGATFALSE 2 GCTGCTTGCGAT 4883 (1:10)   924 ACCAATAG ACCAATAG pLas + pR_LG20170 T1 C03 pL42_ 96.20% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5307 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 C03 sgRNA 89.60% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 5020 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 C04 pL42_ 96.90% CAACATCCTGG FALSE 2 AGAGCAGAAAGG 5495 (1:10)  924 pool1 GGCACAAGC pLas + pR_LG 20170 T1 C04 sgRNA 88.30% CAACATCCTGGFALSE 2 CAACATCCTGGG 4555 (1:10)   924 GGCACAAGC GCACAAGC pLas + pR_LG20170 T1 C05 pL42_ 97.10% GCCGTGCCGTA FALSE 2 AGATGATAACGG 5613 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 C05 sgRNA 88.20% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 4590 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 C06 pL42_ 97.10% GGACGCTAAAC FALSE 2 TCCAGAGCACCT 6229 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 C06 sgRNA 89.80% GGACGCTAAACFALSE 2 GGACGCTAAACC 4789 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 C07 pL42_ 96.50% AGGAGGACGGC FALSE 2 AGCGACCTTACA 5522 (1:10)  924 pool1 AACATCCTG pLas + pR_LG 20170 T1 C07 sgRNA 90.50% AGGAGGACGGCFALSE 2 AGGAGGACGGCA 5621 (1:10)   924 AACATCCTG ACATCCTG pLas + pR_LG20170 T1 C08 pL42_ 96.70% GCTGCTTGCGAT FALSE 2 AGCAGTGTACCT 5399 (1:10)  924 pool1 ACCAATAG pLas + pR_LG 20170 T1 C08 sgRNA 87.90% GCTGCTTGCGATFALSE 2 GCTGCTTGCGAT 3869 (1:10)   924 ACCAATAG ACCAATAG pLas + pR_LG20170 T1 C09 pL42_ 96.60% GCCGTGCCGTA FALSE 2 AGATGATAACGG 5137 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 C09 sgRNA 89.50% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 3866 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 C10 pL42_ 97.30% TGTACTCCAGCT FALSE 2 AGCTAGCCTAGG 6254 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 C10 sgRNA 89.90% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4656 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 C11 pL42_ 97.20% TGTACTCCAGCT FALSE 2 AGCTAGCCTAGG 5845 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 C11 sgRNA 89.60% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4302 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 C12 pL42_ 96.80% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5299 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 C12 sgRNA 86.90% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 3506 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 D01 pL42_ 96.30% GGACGCTAAAC FALSE 2 AGATCGACCACC 5164 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 D01 sgRNA 90.90% GGACGCTAAACFALSE 2 GGACGCTAAACC 4652 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 D02 pL42_ 95.90% CAAGGAGGACG FALSE 2 CTATATATGACC  519 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 D02 sgRNA 94.10% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 5149 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 D03 pL42_ 97.10% GTCCGTTCGACA FALSE 2 CTCAATTTACAG 5103 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 D03 sgRNA 88.70% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4837 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 D04 pL42_ 97.30% TGTACTCCAGCT FALSE 2 AGCGCCGTCATT 5431 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 D04 sgRNA 90.80% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 5191 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 D05 pL42_ 96.90% GCTGCTTGCGAT FALSE 2 GAAGTGGGCAAC 5282 (1:10)  924 pool1 ACCAATAG pLas + pR_LG 20170 T1 D05 sgRNA 90.70% GCTGCTTGCGATFALSE 2 GCTGCTTGCGAT 4882 (1:10)   924 ACCAATAG ACCAATAG pLas + pR_LG20170 T1 D06 pL42_ 97.40% TGTACTCCAGCT FALSE 2 GAACTAGCCACT 6051 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 D06 sgRNA 90.70% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4812 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 D07 pL42_ 97.90% TGTACTCCAGCT FALSE 2 AGCTAGCCTAGG 6191 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 D07 sgRNA 90.60% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4713 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 D08 pL42_ 95.60% GGACGCTAAAC FALSE 2 AGATCGACCACC 5062 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 D08 sgRNA 91.10% GGACGCTAAACFALSE 2 GGACGCTAAACC 4437 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 D10 pL42_ 97.00% GTACAGCTAAG FALSE 2 AGCACGGAGACA 4601 (1:10)  924 pool1 TTAAACTCG pLas + pR_LG 20170 T1 D10 sgRNA 91.70% GTACAGCTAAGFALSE 2 GTACAGCTAAGT 4341 (1:10)   924 TTAAACTCG TAAACTCG pLas + pR_LG20170 T1 D11 pL42_ 97.40% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5112 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 D11 sgRNA 88.80% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 3948 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 D12 pL42_ 97.40% GCCGTGCCGTA FALSE 2 AGATGATAACGG 5286 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 D12 sgRNA 91.20% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 3873 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 E01 pL42_ 97.50% GCCGTGCCGTA FALSE 2 AGATGATAACGG 5806 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 E01 sgRNA 88.40% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 4418 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 E02 pL42_ 96.70% CAAGGAGGACG FALSE 2 CTATATATGACC 4937 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 E02 sgRNA 92.60% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 4515 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 E03 pL42_ 96.30% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5638 (1:10)  924 pool1 GCTATCCGG pLas + pR_LG 20170 T1 E03 sgRNA 90.40% GCCGTGCCGTAFALSE 2 GCCGTGCCGTAG 4364 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 E04 pL42_ 96.90% GTCCGTTCGACA FALSE 2 CTCAATTTACAG 5383 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 E04 sgRNA 87.50% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4141 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 E05 pL42_ 97.30% GGACGCTAAAC FALSE 2 GACAGGCTACCT 5329 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 E05 sgRNA 88.30% GGACGCTAAACFALSE 2 GGACGCTAAACC 4383 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 E06 pL42_ 97.40% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5458 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 E06 sgRNA 88.70% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4981 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 E07 pL42_ 97.00% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5252 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 E07 sgRNA 88.20% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4925 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 E08 pL42_ 96.60% GTACAGCTAAG FALSE 2 AGCACGGAGACA 5176 (1:10)  924 pool1 TTAAACTCG pLas + pR_LG 20170 T1 E08 sgRNA 91.50% GTACAGCTAAGFALSE 2 GTACAGCTAAGT 4568 (1:10)   924 TTAAACTCG TAAACTCG pLas + pR_LG20170 T1 E09 pL42_ 97.00% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5147 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 E09 sgRNA 87.80% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 3963 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 E10 pL42_ 97.10% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 4793 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 E10 sgRNA 88.00% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4321 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 E11 pL42_ 97.50% CAAGGAGGACG FALSE 2 GAACTCAGGACA 4387 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 E11 sgRNA 91.10% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 4034 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 F01 pL42_ 97.20% CAAGGAGGACG FALSE 2 CTATATATGACC 5619 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 F01 sgRNA 89.10% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 2595 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 F02 pL42_ 96.80% CAAGGAGGACG FALSE 2 GAACTCAGGACA 5330 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 F02 sgRNA 92.50% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 3447 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 F03 pL42_ 97.00% CAACATCCTGG FALSE 2 AGATTCATGACG 5301 (1:10)  924 pool1 GGCACAAGC pLas + pR_LG 20170 T1 F03 sgRNA 89.20% CAACATCCTGGFALSE 2 CAACATCCTGGG 4453 (1:10)   924 GGCACAAGC GCACAAGC pLas + pR_LG20170 T1 F04 pL42_ 97.60% TGTACTCCAGCT FALSE 2 AGCGCCGTCATT 5303 (1:10)  924 pool1 TGTGCCCC pLas + pR_LG 20170 T1 F04 sgRNA 89.70% TGTACTCCAGCTFALSE 2 TGTACTCCAGCTT 4379 (1:10)   924 TGTGCCCC GTGCCCC pLas + pR_LG20170 T1 F05 pL42_ 96.70% GTCCGTTCGACA FALSE 2 AGCAACTTCACT 5379 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 F05 sgRNA 86.80% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4602 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 F06 pL42_ 97.10% GTCCGTTCGACA FALSE 2 AGATGGGTTCCG 5479 (1:10)  924 pool1 ATTTCACA pLas + pR_LG 20170 T1 F06 sgRNA 88.40% GTCCGTTCGACAFALSE 2 GTCCGTTCGACA 4357 (1:10)   924 ATTTCACA ATTTCACA pLas + pR_LG20170 T1 F07 pL42_ 97.20% GCTGCTTGCGAT TRUE 2 AGAGCTGCTACG 5788 (1:10)  924 pool1 ACCAATAG pLas + pR_LG 20170 T1 F07 sgRNA 88.60% GCCGTGCCGTATRUE 2 GCCGTGCCGTAG 4696 (1:10)   924 GCTATCCGG CTATCCGG pLas + pR_LG20170 T1 F08 pL42_ 96.70% AAGGAGGACGG FALSE 2 TCCAAAGAGACA 5231 (1:10)  924 pool1 CAACATCCT pLas + pR_LG 20170 T1 F08 sgRNA 90.40% AAGGAGGACGGFALSE 2 AAGGAGGACGGC 4692 (1:10)   924 CAACATCCT AACATCCT pLas + pR_LG20170 T1 F09 pL42_ 97.00% CAAGGAGGACG FALSE 2 AGCCACCAGTAT 5024 (1:10)  924 pool1 GCAACATCC pLas + pR_LG 20170 T1 F09 sgRNA 91.50% CAAGGAGGACGFALSE 2 CAAGGAGGACGG 3869 (1:10)   924 GCAACATCC CAACATCC pLas + pR_LG20170 T1 F10 pL42_ 95.90% GGACGCTAAAC FALSE 2 TCCAGAGCACCT 5167 (1:10)  924 pool1 CAACGGTGC pLas + pR_LG 20170 T1 F10 sgRNA 89.70% GGACGCTAAACFALSE 2 GGACGCTAAACC 4575 (1:10)   924 CAACGGTGC AACGGTGC pLas + pR_LG20170 T1 F11 pL42_ 97.00% GCTGCTTGCGAT FALSE 2 AGCAGTGTACCT 5144 (1:10)  924 pool1 ACCAATAG pLas + pR_LG 20170 T1 F11 sgRNA 89.10% GCTGCTTGCGATFALSE 2 GCTGCTTGCGAT 4141 (1:10)   924 ACCAATAG ACCAATAG pLas + pR_LG20170 T1 F12 pL42_ 96.40% GTACAGCTAAG FALSE 2 GAATTAGTGACC 5578 (1:10)  924 pool1 TTAAACTCG pLas + pR_LG 20170 T1 F12 sgRNA 88.80% GTACAGCTAAGFALSE 2 GTACAGCTAAGT 3825 (1:10)   924 TTAAACTCG TAAACTCG pLas + 20170T2 A01 pL42_ 97.40% TGTACTCCAGCT FALSE 2 AGCGCCGTCATT 5888 pLX_TRC313_  924 pool1 TGTGCCCC LacZ (1:1000) pLas + 20170 T2 A01 sgRNA 90.00%TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 4612 pLX_TRC313_   924 TGTGCCCCGTGCCCC LacZ (1:1000) pLas + 20170 T2 A02 pL42_ 98.10% TGTACTCCAGCTFALSE 2 AGCTAGCCTAGG 6329 pLX_TRC313_   924 pool1 TGTGCCCC LacZ (1:1000)pLas + 20170 T2 A02 sgRNA 91.60% TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 3870pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000) pLas + 20170 T2 A05pL42_ 96.60% TGTACTCCAGCT FALSE 2 AGCTCTGACACA 6816 pLX_TRC313_   924pool1 TGTGCCCC LacZ (1:1000) pLas + 20170 T2 A05 sgRNA 90.10%TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 3081 pLX_TRC313_   924 TGTGCCCCGTGCCCC LacZ (1:1000) pLas + 20170 T2 A06 pL42_ 96.70% TGTACTCCAGCTFALSE 2 CATGATCCCACA 5634 pLX_TRC313_   924 pool1 TGTGCCCC LacZ (1:1000)pLas + 20170 T2 A06 sgRNA 91.10% TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 3763pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000) pLas + 20170 T2 A07pL42_ 96.10% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5778 pLX_TRC313_   924pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 A07 sgRNA 89.40%GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3233 pLX_TRC313_   924 GCTATCCGGCTATCCGG LacZ (1:1000) pLas + 20170 T2 A08 pL42_ 96.40% AAGGAGGACGGFALSE 2 GAATGAACCACG 5705 pLX_TRC313_   924 pool1 CAACATCCTLacZ (1:1000) pLas + 20170 T2 A08 sgRNA 91.70% AAGGAGGACGG FALSE 2AAGGAGGACGGC 2902 pLX_TRC313_   924 CAACATCCT AACATCCT LacZ (1:1000)pLas +   20170 T2 A09 pL42_ 97.40% CAAGGAGGACG FALSE 2 CTATATATGACC 4537pLX_TRC313_   924 pool1 GCAACATCC LacZ (1:1000) pLas + 20170 T2 A09sgRNA 92.00% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 2869 pLX_TRC313_   924GCAACATCC CAACATCC LacZ (1:1000) pLas + 20170 T2 A10 pL42_ 97.00%TGTACTCCAGCT FALSE 2 GAACTAGCCACT 6008 pLX_TRC313_   924 pooll TGTGCCCCLacZ (1:1000) pLas + 20170 T2 A10 sgRNA 90.70% TGTACTCCAGCT FALSE 2TGTACTCCAGCTT 3110 pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000)pLas + 20170 T2 A11 pL42_ 97.10% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5570pLX_TRC313_   924 pooll ATTTCACA LacZ (1:1000) pLas + 20170 T2 A11 sgRNA86.90% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 3042 pLX_TRC313_   924 ATTTCACAATTTCACA LacZ (1:1000) pLas + 20170 T2 A12 pL42_ 97.70% GCTGCTTGCGATFALSE 2 AGCAGCCCTAGC 5860 pLX_TRC313_   924 pooll ACCAATAG LacZ (1:1000)pLas + 20170 T2 A12 sgRNA 89.60% GCTGCTTGCGAT FALSE 2 GCTGCTTGCGAT 2508pLX_TRC313_   924 ACCAATAG ACCAATAG LacZ (1:1000) pLas + 20170 T2 B01pL42_ 96.20% AGGAGGACGGC FALSE 2 AGCGACCTTACA 5516 pLX_TRC313_   924pooll AACATCCTG LacZ (1:1000) pLas + 20170 T2 B01 sgRNA 91.00%AGGAGGACGGC FALSE 2 AGGAGGACGGCA 3684 pLX_TRC313_   924 AACATCCTGACATCCTG LacZ (1:1000) pLas + 20170 T2 B02 pL42_ 96.20% CAAGGAGGACGFALSE 2 GAACTCAGGACA 6017 pLX_TRC313_   924 pooll GCAACATCCLacZ (1:1000) pLas + 20170 T2 B02 sgRNA 92.90% CAAGGAGGACG FALSE 2CAAGGAGGACGG 3527 pLX_TRC313_   924 GCAACATCC CAACATCC LacZ (1:1000)pLas + 20170 T2 B03 pL42_ 97.30% GTCCGTTCGACA FALSE 2 GACAAGTACACT 5069pLX_TRC313_   924 pooll ATTTCACA LacZ (1:1000) pLas + 20170 T2 B03 sgRNA88.00% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 3034 pLX_TRC313_   924 ATTTCACAATTTCACA LacZ (1:1000) pLas + 20170 T2 B04 pL42_ 96.60% AGGAGGACGGCFALSE 2 AGCGACCTTACA 5231 pLX_TRC313_   924 pool1 AACATCCTGLacZ (1:1000) pLas + 20170 T2 B04 sgRNA 90.20% AGGAGGACGGC FALSE 2AGGAGGACGGCA 3211 pLX_TRC313_   924 AACATCCTG ACATCCTG LacZ (1:1000)pLas + 20170 T2 B05 pL42_ 97.30% GCCGTGCCGTA FALSE 2 GACATGCGTAGC 5816pLX_TRC313_   924 pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 B05sgRNA 89.70% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3329 pLX_TRC313_   924GCTATCCGG CTATCCGG LacZ (1:1000) pLas + 20170 T2 B06 pL42_ 95.70%GGACGCTAAAC FALSE 2 AGATCGACCACC 5445 pLX_TRC313_   924 pool1 CAACGGTGCLacZ (1:1000) pLas + 20170 T2 B06 sgRNA 90.40% GGACGCTAAAC FALSE 2GGACGCTAAACC 2914 pLX_TRC313_   924 CAACGGTGC AACGGTGC LacZ (1:1000)pLas + 20170 T2 B07 pL42_ 96.30% AAGGAGGACGG FALSE 2 GAATGAACCACG 5476pLX_TRC313_   924 pool1 CAACATCCT LacZ (1:1000) pLas + 20170 T2 B07sgRNA 90.40% AAGGAGGACGG FALSE 2 AAGGAGGACGGC 3405 pLX_TRC313_   924CAACATCCT AACATCCT LacZ (1:1000) pLas + 20170 T2 B08 pL42_ 96.70%TGTACTCCAGCT FALSE 2 AGCTCTGACACA 5351 pLX_TRC313_   924 pool1 TGTGCCCCLacZ (1:1000) pLas + 20170 T2 B08 sgRNA 90.90% TGTACTCCAGCT FALSE 2TGTACTCCAGCTT 3306 pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000)pLas + 20170 T2 B09 pL42_ 97.40% GTACAGCTAAG FALSE 2 TCCATCAATACG 5332pLX_TRC313_   924 pool1 TTAAACTCG LacZ (1:1000) pLas + 20170 T2 B09sgRNA 90.30% GTACAGCTAAG FALSE 2 GTACAGCTAAGT 3539 pLX_TRC313_   924TTAAACTCG TAAACTCG LacZ (1:1000) pLas + 20170 T2 B10 pL42_ 96.80%GGACGCTAAAC FALSE 2 GACAGGCTACCT 5495 pLX_TRC313_   924 pool1 CAACGGTGCLacZ (1:1000) pLas + 20170 T2 B10 sgRNA 90.90% GGACGCTAAAC FALSE 2GGACGCTAAACC 3426 pLX_TRC313_   924 CAACGGTGC AACGGTGC LacZ (1:1000)pLas + 20170 T2 B11 pL42_ 96.20% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5434pLX_TRC313_   924 pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 B11sgRNA 90.00% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 2941 pLX_TRC313_   924GCTATCCGG CTATCCGG LacZ (1:1000) pLas + 20170 T2 B12 pL42_ 96.30%AGGAGGACGGC FALSE 2 AGCGACCTTACA 5569 pLX_TRC313_   924 pool1 AACATCCTGLacZ (1:1000) pLas + 20170 T2 B12 sgRNA 91.10% AGGAGGACGGC FALSE 2AGGAGGACGGCA 2966 pLX_TRC313_   924 AACATCCTG ACATCCTG LacZ (1:1000)pLas + 20170 T2 C01 pL42_ 97.70% GTACAGCTAAG FALSE 2 CTAGATGTTAGC 6135pLX_TRC313_   924 pool1 TTAAACTCG LacZ (1:1000) pLas + 20170 T2 C01sgRNA 88.80% GTACAGCTAAG FALSE 2 GTACAGCTAAGT 3119 pLX_TRC313_   924TTAAACTCG TAAACTCG LacZ (1:1000) pLas + 20170 T2 C02 pL42_ 97.10%TGTACTCCAGCT FALSE 2 AGCGCCGTCATT 5592 pLX_TRC313_   924 pool1 TGTGCCCCLacZ (1:1000) pLas + 20170 T2 C02 sgRNA 90.60% TGTACTCCAGCT FALSE 2TGTACTCCAGCTT 3382 pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000)pLas + 20170 T2 C03 pL42_ 96.90% GCCGTGCCGTA FALSE 2 GACAACGAGAAC 5672pLX_TRC313_   924 pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 C03sgRNA 90.70% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3822 pLX_TRC313_   924GCTATCCGG CTATCCGG LacZ (1:1000) pLas + 20170 T2 C04 pL42_ 97.20%AGGAGGACGGC FALSE 2 GAATGGACAGCG 5859 pLX_TRC313_   924 pool1 AACATCCTGLacZ (1:1000) pLas + 20170 T2 C04 sgRNA 91.00% AGGAGGACGGC FALSE 2AGGAGGACGGCA 3447 pLX_TRC313_   924 AACATCCTG ACATCCTG LacZ (1:1000)pLas + 20170 T2 C05 pL42_ 97.30% CAACATCCTGG FALSE 2 GAACGCGAAAGC 5788pLX_TRC313_   924 pool1 GGCACAAGC LacZ (1:1000) pLas + 20170 T2 C05sgRNA 89.70% CAACATCCTGG FALSE 2 CAACATCCTGGG 3877 pLX_TRC313_   924GGCACAAGC GCACAAGC LacZ (1:1000) pLas + 20170 T2 C06 pL42_ 96.90%GTCCGTTCGACA FALSE 2 GACAAGTACACT 6389 pLX_TRC313_   924 pool1 ATTTCACALacZ (1:1000) pLas + 20170 T2 C06 sgRNA 87.60% GTCCGTTCGACA FALSE 2GTCCGTTCGACA 3539 pLX_TRC313_   924 ATTTCACA ATTTCACA LacZ (1:1000)pLas + 20170 T2 C09 pL42_ 96.10% GCCGTGCCGTA FALSE 2 CTCACTGACACT 5733pLX_TRC313_   924 pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 C09sgRNA 90.70% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3107 pLX_TRC313_   924GCTATCCGG CTATCCGG LacZ (1:1000) pLas + 20170 T2 C11 pL42_ 96.50%AAGGAGGACGG FALSE 2 TCCAAAGAGACA 5408 pLX_TRC313_   924 pool1 CAACATCCTLacZ (1:1000) pLas + 20170 T2 C11 sgRNA 91.40% AAGGAGGACGG FALSE 2AAGGAGGACGGC 3554 pLX_TRC313_   924 CAACATCCT AACATCCT LacZ (1:1000)pLas + 20170 T2 C12 pL42_ 96.40% GGACGCTAAAC FALSE 2 TCCAGAGCACCT 6109pLX_TRC313_   924 pool1 CAACGGTGC LacZ (1:1000) pLas + 20170 T2 C12sgRNA 89.80% GGACGCTAAAC FALSE 2 GGACGCTAAACC 3255 pLX_TRC313_   924CAACGGTGC AACGGTGC LacZ (1:1000) pLas + 20170 T2 D01 pL42_ 96.90%GGACGCTAAAC FALSE 2 AGAGACTTCACA 5626 pLX_TRC313_   924 pool1 CAACGGTGCLacZ (1:1000) pLas + 20170 T2 D01 sgRNA 88.60% GGACGCTAAAC FALSE 2GGACGCTAAACC 2686 pLX_TRC313_   924 CAACGGTGC AACGGTGC LacZ (1:1000)pLas + 20170 T2 D02 pL42_ 96.80% GGACGCTAAAC FALSE 2 TCCAGAGCACCT 6040pLX_TRC313_   924 pool1 CAACGGTGC LacZ (1:1000) pLas + 20170 T2 D02sgRNA 91.20% GGACGCTAAAC FALSE 2 GGACGCTAAACC 3286 pLX_TRC313_   924CAACGGTGC AACGGTGC LacZ (1:1000) pLas + 20170 T2 D03 pL42_ 97.10%GCTGCTTGCGAT TRUE 2 GAAGTGGGCAAC 5323 pLX_TRC313_   924 pool1 ACCAATAGLacZ (1:1000) pLas + 20170 T2 D03 sgRNA 90.30% TGTACTCCAGCT TRUE 2TGTACTCCAGCTT 3042 pLX_TRC313_   924 TGTGCCCC GTGCCCC LacZ (1:1000)pLas + 20170 T2 D04 pL42_ 96.30% TGTACTCCAGCT FALSE 2 AGCTCTGACACA 5512pLX_TRC313_   924 pool1 TGTGCCCC LacZ (1:1000) pLas + 20170 T2 D04 sgRNA89.40% TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 2991 pLX_TRC313_   924TGTGCCCC GTGCCCC LacZ (1:1000) pLas + 20170 T2 D05 pL42_ 96.20%GCCGTGCCGTA FALSE 2 CTCACTGACACT 5837 pLX_TRC313_   924 pool1 GCTATCCGGLacZ (1:1000) pLas + 20170 T2 D05 sgRNA 89.70% GCCGTGCCGTA FALSE 2GCCGTGCCGTAG 3416 pLX_TRC313_   924 GCTATCCGG CTATCCGG LacZ (1:1000)pLas + 20170 T2 D06 pL42_ 98.00% TGTACTCCAGCT FALSE 2 AGCTAGCCTAGG 6296pLX_TRC313_   924 pool1 TGTGCCCC LacZ (1:1000) pLas + 20170 T2 D06 sgRNA90.50% TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 3879 pLX_TRC313_   924TGTGCCCC GTGCCCC LacZ (1:1000) pLas + 20170 T2 D07 pL42_ 96.80%AAGGAGGACGG FALSE 2 CTAGTGTCCACA 5837 pLX_TRC313_   924 pool1 CAACATCCTLacZ (1:1000) pLas + 20170 T2 D07 sgRNA 91.60% AAGGAGGACGG FALSE 2AAGGAGGACGGC 3677 pLX_TRC313_   924 CAACATCCT AACATCCT LacZ (1:1000)pLas + 20170 T2 D08 pL42_ 97.10% CAAGGAGGACG FALSE 2 AGCCAGTCAATC 6163pLX_TRC313_   924 pool1 GCAACATCC LacZ (1:1000) pLas + 20170 T2 D08sgRNA 90.60% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 3415 pLX_TRC313_   924GCAACATCC CAACATCC LacZ (1:1000) pLas + 20170 T2 D09 pL42_ 97.50%GCCGTGCCGTA TRUE 2 GACATGCGTAGC 5736 pLX_TRC313_   924 pool1 GCTATCCGGLacZ (1:1000) pLas + 20170 T2 D09 sgRNA 88.00% GTCCGTTCGACA TRUE 2GTCCGTTCGACA 3192 pLX_TRC313_   924 ATTTCACA ATTTCACA LacZ (1:1000)pLas + 20170 T2 D10 pL42_ 45.70% GCCGTGCCGTA FALSE 4 AGATGATAACGG 2661pLX_TRC313_   924 pool1 GCTATCCGG LacZ (1:1000) pLas + 20170 T2 D10pL42_ 51.10% GTCCGTTCGACA FALSE 4 AGCAACTTCACT 2976 pLX_TRC313_   924pool1 ATTTCACA LacZ (1:1000) pLas + 20170 T2 D10 sgRNA 43.30%GCCGTGCCGTA FALSE 4 GCCGTGCCGTAG 1374 pLX_TRC313_   924 GCTATCCGGCTATCCGG LacZ (1:1000) pLas + 20170 T2 D10 sgRNA 44.70% GTCCGTTCGACAFALSE 4 GTCCGTTCGACA 1416 pLX_TRC313_   924 ATTTCACA ATTTCACALacZ (1:1000) pLas + 20170 T2 D11 pL42_ 96.60% GTCCGTTCGACA FALSE 2AGCAACTTCACT 5158 pLX_TRC313_   924 pool1 ATTTCACA LacZ (1:1000) pLas +20170 T2 D11 sgRNA 86.80% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 2929pLX_TRC313_   924 ATTTCACA ATTTCACA LacZ (1:1000) pLas + 20170 T2 D12pL42_ 97.30% GCTGCTTGCGAT FALSE 2 AGCAGCCCTAGC 5908 pLX_TRC313_   924pool1 ACCAATAG LacZ (1:1000) pLas + 20170 T2 D12 sgRNA 89.00%GCTGCTTGCGAT FALSE 2 GCTGCTTGCGAT 3297 pLX_TRC313_   924 ACCAATAGACCAATAG LacZ (1:1000) pLas + 20170 T2 E01 pL42_ 97.50% CAAGGAGGACGFALSE 2 GAACTCAGGACA 6341 pLX_TRC313_   924 pool1 GCAACATCCLacZ (1:1000) pLas + 20170 T2 E01 sgRNA 90.10% CAAGGAGGACG FALSE 2CAAGGAGGACGG 3297 pLX_TRC313_   924 GCAACATCC CAACATCC LacZ (1:1000)pLas + 20170 T2 E02 pL42_ 96.60% CAAGGAGGACG FALSE 2 CTATATATGACC 5665pLX_TRC313_   924 pool1 GCAACATCC LacZ (1:1000) pLas + 20170 T2 E02sgRNA 92.20% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 3177 pLX_TRC313_   924GCAACATCC CAACATCC LacZ (1:1000) pLas + 20170 T2 E03 pL42_ 96.00%GTCCGTTCGACA FALSE 2 CTCAATTTACAG 5694 pLX_TRC313_   924 pool1 ATTTCACALacZ (1:1000) pLas + 20170 T2 E03 sgRNA 86.90% GTCCGTTCGACA FALSE 2GTCCGTTCGACA 2666 pLX_TRC313_   924 ATTTCACA ATTTCACA LacZ (1:1000)pLas + 20170 T2 E04 pL42_ 96.20% GGACGCTAAAC FALSE 2 TCCAGAGCACCT 5711pLX_TRC313_   924 pool1 CAACGGTGC LacZ (1:1000) pLas + 20170 T2 E04sgRNA 88.20% GGACGCTAAAC FALSE 2 GGACGCTAAACC 3074 pLX_TRC313_   924CAACGGTGC AACGGTGC LacZ (1:1000) pLas + 20170 T2 E05 pL42_ 96.10%GGACGCTAAAC FALSE 2 AGAGACTTCACA 6920 pLX_TRC313_   924 pool1 CAACGGTGCLacZ (1:1000) pLas + 20170 T2 E05 sgRNA 88.80% GGACGCTAAAC FALSE 2GGACGCTAAACC 3316 pLX_TRC313_   924 CAACGGTGC AACGGTGC LacZ (1:1000)pLas + 20170 T2 E06 pL42_ 96.50% CAAGGAGGACG FALSE 2 AGCCACCAGTAT 5948pLX_TRC313_   924 pool1 GCAACATCC LacZ (1:1000) pLas + 20170 T2 E06sgRNA 90.70% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 3701 pLX_TRC313_   924GCAACATCC CAACATCC LacZ (1:1000) pLas + 20170 T2 E07 pL42_ 96.90%CAACATCCTGG FALSE 2 GAACGCGAAAGC 6218 pLX_TRC313_   924 pool1 GGCACAAGCLacZ (1:1000) pLas + 20170 T2 E07 sgRNA 88.20% CAACATCCTGG FALSE 2CAACATCCTGGG 3216 pLX_TRC313_   924 GGCACAAGC GCACAAGC LacZ (1:1000)pLas + 20170 T2 E09 pL42_ 97.30% TGTACTCCAGCT FALSE 2 AGCTAGCCTAGG 6260pLX_TRC313_   924 pool1 TGTGCCCC LacZ (1:1000) pLas + 20170 T2 E09 sgRNA89.40% TGTACTCCAGCT FALSE 2 TGTACTCCAGCTT 2857 pLX_TRC313_   924TGTGCCCC GTGCCCC LacZ (1:1000) pLas + 20170 T2 E10 pL42_ 95.60%AGGAGGACGGC FALSE 2 AGCGACCTTACA 5526 pLX TRC313   924 pool1 AACATCCTGLacZ (1:1000) pLas + 20170 T2 E10 sgRNA 90.80% AGGAGGACGGC FALSE 2AGGAGGACGGCA 3033 pLX TRC313   924 AACATCCTG ACATCCTG LacZ (1:1000)pLas + 20170 T2 E11 pL42_ 97.20% AAGGAGGACGG FALSE 2 GAATCCGCTCGC 5387pLX TRC313   924 pool1 CAACATCCT LacZ (1:1000) pLas + 20170 T2 E11 sgRNA91.20% AAGGAGGACGG FALSE 2 AAGGAGGACGGC 2772 pLX TRC313   924 CAACATCCTAACATCCT LacZ (1:1000) pLas, 20170 T2 F01 pL42_ 97.50% GCCGTGCCGTA FALSE2 AGATGATAACGG 6077 standard_400   924 pool1 GCTATCCGG pLas, 20170 T2F01 sgRNA 88.50% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3723 standard_400  924 GCTATCCGG CTATCCGG pLas, 20170 T2 F02 pL42_ 97.50% CAAGGAGGACGFALSE 2 AGCCACCAGTAT 5535 standard_400   924 pool1 GCAACATCC pLas, 20170T2 F02 sgRNA 91.70% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 3850 standard_400  924 GCAACATCC CAACATCC pLas, 20170 T2 F04 pL42_ 96.80% GTACAGCTAAGTRUE 2 TCCATCAATACG 5844 standard_400   924 pool1 TTAAACTCG pLas, 20170T2 F04 sgRNA 90.30% CAAGGAGGACG TRUE 2 CAAGGAGGACGG 3878 standard_400  924 GCAACATCC CAACATCC pLas, 20170 T2 F05 pL42_ 96.90% GTCCGTTCGACAFALSE 2 AGATGGGTTCCG 5399 standard_400   924 pool1 ATTTCACA pLas, 20170T2 F05 sgRNA 86.50% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 3780 standard_400  924 ATTTCACA ATTTCACA pLas, 20170 T2 F06 pL42_ 96.20% GCCGTGCCGTAFALSE 2 CTCACTGACACT 6156 standard_400   924 pool1 GCTATCCGG pLas, 20170T2 F06 sgRNA 90.90% GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 3688 standard_400  924 GCTATCCGG CTATCCGG pLas, 20170 T2 F07 pL42_ 97.00% GTCCGTTCGACAFALSE 2 AGATGGGTTCCG 6019 standard_400   924 pool1 ATTTCACA pLas, 20170T2 F07 sgRNA 88.60% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 3235 standard_400  924 ATTTCACA ATTTCACA pLas, 20170 T2 G01 pL42_ 46.40% GTCCGTTCGACAFALSE 4 AGCAACTTCACT 2689 standard_400   924 pool1 ATTTCACA pLas, 20170T2 G01 pL42_ 50.90% TGTACTCCAGCT TRUE 4 AGCTAGCCTAGG 2953 standard_400  924 pool1 TGTGCCCC pLas, 20170 T2 G01 sgRNA 40.80% GTCCGTTCGACA FALSE4 GTCCGTTCGACA 1683 standard_400   924 ATTTCACA ATTTCACA pLas, 20170 T2G01 sgRNA 41.40% CAAGGAGGACG TRUE 4 CAAGGAGGACGG 1711 standard_400   924GCAACATCC CAACATCC pLas, 20170 T2 G02 pL42_ 97.00% GTCCGTTCGACA FALSE 2GACAAGTACACT 5757 standard_400   924 pool1 ATTTCACA pLas, 20170 T2 G02sgRNA 87.80% GTCCGTTCGACA FALSE 2 GTCCGTTCGACA 3507 standard_400   924ATTTCACA ATTTCACA pLas, 20170 T2 G03 pL42_ 96.80% GTCCGTTCGACA TRUE 2AGATGGGTTCCG 5176 standard_400   924 pool1 ATTTCACA pLas, 20170 T2 G03sgRNA 90.90% AAGGAGGACGG TRUE 2 AAGGAGGACGGC 3746 standard_400   924CAACATCCT AACATCCT pLas, 20170 T2 G04 pL42_ 97.00% CAAGGAGGACG TRUE 2AGCCAGTCAATC 8030 standard_400   924 pool1 GCAACATCC pLas, 20170 T2 G04sgRNA 86.70% GTCCGTTCGACA TRUE 2 GTCCGTTCGACA 3434 standard_400   924ATTTCACA ATTTCACA pLas, 20170 T2 G05 pL42_ 96.80% GTCCGTTCGACA TRUE 2AGCAACTTCACT 5624 standard_400   924 pool1 ATTTCACA pLas, 20170 T2 G05sgRNA 90.20% CAACATCCTGG TRUE 2 CAACATCCTGGG 4140 standard_400   924GGCACAAGC GCACAAGC pLas, 20170 T2 G06 pL42_ 44.90% GGACGCTAAAC FALSE 4GACAGGCTACCT 2487 standard_400   924 pool1 CAACGGTGC pLas, 20170 T2 G06pL42_ 52.20% AGGAGGACGGC FALSE 4 GAATGGACAGCG 2895 standard_400   924pool1 AACATCCTG pLas, 20170 T2 G06 sgRNA 44.80% GGACGCTAAAC FALSE 4GGACGCTAAACC 1766 standard_400   924 CAACGGTGC AACGGTGC pLas, 20170 T2G06 sgRNA 46.10% AGGAGGACGGC FALSE 4 AGGAGGACGGCA 1817 standard_400  924 AACATCCTG ACATCCTG pLas, 20170 T2 G07 pL42_ 97.30% TGTACTCCAGCTTRUE 2 AGCGCCGTCATT 5570 standard_400   924 pool1 TGTGCCCC pLas, 20170T2 G07 sgRNA 89.90% AAGGAGGACGG TRUE 2 AAGGAGGACGGC 3693 standard_400  924 CAACATCCT AACATCCT pLas, 20170 T2 G08 pL42_ 96.60% TGTACTCCAGCTTRUE 2 AGCGCCGTCATT 5248 standard_400   924 pool1 TGTGCCCC pLas, 20170T2 G08 sgRNA 91.60% AAGGAGGACGG TRUE 2 AAGGAGGACGGC 3721 standard_400  924 CAACATCCT AACATCCT pLas, 20170 T2 G09 pL42_ 47.40% AAGGAGGACGGTRUE 4 GAATGAACCACG 2772 standard_400   924 pool1 CAACATCCT pLas, 20170T2 G09 pL42_ 49.50% CAACATCCTGG FALSE 4 GAACGCGAAAGC 2895 standard_400  924 pool1 GGCACAAGC pLas, 20170 T2 G09 sgRNA 43.30% CAACATCCTGG FALSE4 CAACATCCTGGG 1706 standard_400   924 GGCACAAGC GCACAAGC pLas, 20170 T2G09 sgRNA 44.60% GCCGTGCCGTA TRUE 4 GCCGTGCCGTAG 1760 standard_400   924GCTATCCGG CTATCCGG pLas, 20170 T2 G10 pL42_ 96.50% CAAGGAGGACG FALSE 2AGCCAGTCAATC 6342 standard_400   924 pool1 GCAACATCC pLas, 20170 T2 G10sgRNA 91.60% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 3814 standard_400   924GCAACATCC CAACATCC pLas, 20170 T2 G11 pL42_ 97.20% AGGAGGACGGC FALSE 2AGCGGTATAACT 5601 standard_400   924 pool1 AACATCCTG pLas, 20170 T2 G11sgRNA 90.60% AGGAGGACGGC FALSE 2 AGGAGGACGGCA 3625 standard_400   924AACATCCTG ACATCCTG pLas, 20170 T2 G12 pL42_ 97.00% GTACAGCTAAG FALSE 2TCCATCAATACG 6172 standard_400   924 pool1 TTAAACTCG pLas, 20170 T2 G12sgRNA 90.50% GTACAGCTAAG FALSE 2 GTACAGCTAAGT 4107 standard_400   924TTAAACTCG TAAACTCG pLas, 20170 T2 H02 pL42_ 96.60% CAACATCCTGG FALSE 2AGCTTTCTGACT 6904 standard_400   924 pool1 GGCACAAGC pLas, 20170 T2 H02sgRNA 89.70% CAACATCCTGG FALSE 2 CAACATCCTGGG 4167 standard_400   924GGCACAAGC GCACAAGC pLas, 20170 T2 H04 pL42_ 97.10% CAACATCCTGG FALSE 2GAACGCGAAAGC 6597 standard_400   924 pool1 GGCACAAGC pLas, 20170 T2 H04sgRNA 88.90% CAACATCCTGG FALSE 2 CAACATCCTGGG 4441 standard_400   924GGCACAAGC GCACAAGC pLas, 20170 T2 H05 pL42_ 96.90% AGGAGGACGGC FALSE 2AGCGGTATAACT 5605 standard_400   924 pool1 AACATCCTG pLas, 20170 T2 H05sgRNA 91.70% AGGAGGACGGC FALSE 2 AGGAGGACGGCA 5041 standard_400   924AACATCCTG ACATCCTG pLas, 20170 T2 H06 pL42_ 97.20% GCTGCTTGCGAT TRUE 2AGAGCTGCTACG 6005 standard_400   924 pool1 ACCAATAG pLas, 20170 T2 H06sgRNA 90.60% AAGGAGGACGG TRUE 2 AAGGAGGACGGC 4160 standard_400   924CAACATCCT AACATCCT pLas, 20170 T2 H07 pL42_ 97.00% CAAGGAGGACG TRUE 2AGCCAGTCAATC 6670 standard_400   924 pool1 GCAACATCC pLas, 20170 T2 H07sgRNA 91.40% TGTACTCCAGCT TRUE 2 TGTACTCCAGCTT 3692 standard_400   924TGTGCCCC GTGCCCC pLas, 20170 T2 H08 pL42_ 47.30% AAGGAGGACGG TRUE 4GAATGAACCACG 2843 standard_400   924 pool1 CAACATCCT pLas, 20170 T2 H08pL42_ 49.30% CAACATCCTGG FALSE 4 GAACGCGAAAGC 2960 standard_400   924pool1 GGCACAAGC pLas, 20170 T2 H08 sgRNA 42.30% CAACATCCTGG FALSE 4CAACATCCTGGG 1458 standard_400   924 GGCACAAGC GCACAAGC pLas, 20170 T2H08 sgRNA 46.70% GCCGTGCCGTA TRUE 4 GCCGTGCCGTAG 1607 standard_400   924GCTATCCGG CTATCCGG pLas, 20170 T2 H09 pL42_ 97.10% AGGAGGACGGC FALSE 2AGCGGTATAACT 6040 standard_400   924 pool1 AACATCCTG pLas, 20170 T2 H09sgRNA 91.30% AGGAGGACGGC FALSE 2 AGGAGGACGGCA 4335 standard_400   924AACATCCTG ACATCCTG pLas, 20170 T2 H10 pL42_ 96.40% GTACAGCTAAG FALSE 2TCCATCAATACG 6012 standard_400   924 pool1 TTAAACTCG pLas, 20170 T2 H10sgRNA 91.80% GTACAGCTAAG FALSE 2 GTACAGCTAAGT 4678 standard_400   924TTAAACTCG TAAACTCG pLas, 20170 T2 H11 pL42_ 97.20% GGACGCTAAAC FALSE 2GACAGGCTACCT 2525 standard_400   924 pool1 CAACGGTGC pLas, 20170 T2 H11sgRNA 90.50% GGACGCTAAAC FALSE 2 GGACGCTAAACC 4621 standard_400   924CAACGGTGC AACGGTGC pLas, 20170 T2 H12 pL42_ 96.60% GGACGCTAAAC FALSE 2AGAGACTTCACA 6601 standard_400   924 pool1 CAACGGTGC pLas, 20170 T2 H12sgRNA 90.60% GGACGCTAAAC FALSE 2 GGACGCTAAACC 5162 standard_400   924CAACGGTGC AACGGTGC pLas + 20170 T1 G01 pL42_ 96.40% AGGAGGACGGC FALSE 2GATATCGTGACC 5913 pLX TRC313   924 pool1 AACATCCTG LacZ (1:100) pLas +20170 T1 G01 sgRNA 89.90% AGGAGGACGGC FALSE 2 AGGAGGACGGCA 1376pLX TRC313   924 AACATCCTG ACATCCTG LacZ (1:100) pLas + 20170 T1 G02pL42_ 97.00% CAACATCCTGG FALSE 2 CAACGCCCAAGG 5458 pLX TRC313   924pool1 LacZ (1:100) GGCACAAGC pLas + 20170 T1 G02 sgRNA 91.20%CAACATCCTGG FALSE 2 CAACATCCTGGG 4271 pLX TRC313   924 GGCACAAGCGCACAAGC LacZ (1:100) pLas + 20170 T1 G03 pL42_ 46.10% AAGGAGGACGG FALSE4 TAGGAAGTTAGG 2373 pLX TRC313   924 pool1 CAACATCCT LacZ (1:100) pLas +20170 T1 G03 pL42_ 50.90% TGTACTCCAGCT FALSE 4 TTCGCCGAAAGC 2622pLX TRC313   924 pool1 TGTGCCCC LacZ (1:100) pLas + 20170 T1 G03 sgRNA43.60% TGTACTCCAGCT FALSE 4 TGTACTCCAGCTT 2285 pLX TRC313   924 TGTGCCCCGTGCCCC LacZ (1:100) pLas + 20170 T1 G03 sgRNA 47.10% AAGGAGGACGG FALSE4 AAGGAGGACGGC 2470 pLX_TRC313_   924 CAACATCCT AACATCCT LacZ (1:100)pLas + 20170 T1 G04 pL42_ 97.80% CAAGGAGGACG FALSE 2 AGGCTATTAATG 5677pLX_TRC313_   924 pool1 GCAACATCC LacZ (1:100) pLas + 20170 T1 G04 sgRNA90.80% CAAGGAGGACG FALSE 2 CAAGGAGGACGG 4803 pLX_TRC313_   924 GCAACATCCCAACATCC LacZ (1:100) pLas + 20170 T1 G05 pL42_ 97.20% GCTGCTTGCGATFALSE 2 ATCAGTGGCAGC 5807 pLX_TRC313_   924 pool1 ACCAATAG LacZ (1:100)pLas + 20170 T1 G05 sgRNA 89.20% GCTGCTTGCGAT FALSE 2 GCTGCTTGCGAT 4589pLX_TRC313_   924 ACCAATAG ACCAATAG LacZ (1:100) pLas + 20170 T1 G06pL42_ 96.90% CAACATCCTGG TRUE 2 TGTTAATGCAGG 7332 pLX_TRC313_   924pool1 GGCACAAGC LacZ (1:100) pLas + 20170 T1 G06 sgRNA 90.50%GGACGCTAAAC TRUE 2 GGACGCTAAACC 4568 pLX_TRC313_   924 CAACGGTGCAACGGTGC LacZ (1:100) pLas + 20170 T1 G07 pL42_ 96.80% CAACATCCTGG FALSE2 TCTATTTGACGG 5698 pLX_TRC313_   924 pool1 GGCACAAGC LacZ (1:100)pLas + 20170 T1 G07 sgRNA 89.70% CAACATCCTGG FALSE 2 CAACATCCTGGG 3855pLX_TRC313_   924 GGCACAAGC GCACAAGC LacZ (1:100) pLas + 20170 T1 G08pL42_ 95.60% CAACATCCTGG FALSE 2 CCGTAACGAACA 5272 pLX_TRC313_   924pool1 GGCACAAGC LacZ (1:100) pLas + 20170 T1 G08 sgRNA 89.80%CAACATCCTGG FALSE 2 CAACATCCTGGG 3373 pLX_TRC313_   924 GGCACAAGCGCACAAGC LacZ (1:100) pLas + 20170 T1 G09 pL42_ 97.20% GGACGCTAAAC FALSE2 AGTTTGCAGCCA 5286 pLX_TRC313_   924 pool1 CAACGGTGC LacZ (1:100)pLas + 20170 T1 G09 sgRNA 90.80% GGACGCTAAAC FALSE 2 GGACGCTAAACC 3837pLX_TRC313_   924 CAACGGTGC AACGGTGC LacZ (1:100) pLas + 20170 T1 G10pL42_ 95.30% GGACGCTAAAC FALSE 2 TCGAAATGACAC 5229 pLX_TRC313_   924pool1 CAACGGTGC LacZ (1:100) pLas + 20170 T1 G10 sgRNA 90.70%GGACGCTAAAC FALSE 2 GGACGCTAAACC 4325 pLX_TRC313_   924 CAACGGTGCAACGGTGC LacZ (1:100) pLas + 20170 T1 G11 pL42_ 96.80% AGGAGGACGGC FALSE2 TCAGTGAATACG 4733 pLX_TRC313_   924 pool1 AACATCCTG LacZ (1:100)pLas + 20170 T1 G11 sgRNA 90.90% AGGAGGACGGC FALSE 2 AGGAGGACGGCA 3508pLX_TRC313_   924 AACATCCTG ACATCCTG LacZ (1:100) pLas + 20170 T1 G12pL42_ 96.50% GCCGTGCCGTA FALSE 2 CGCAAAAGGATT 5455 pLX_TRC313_   924pool1 GCTATCCGG LacZ (1:100) pLas + 20170 T1 G12 sgRNA 89.20%GCCGTGCCGTA FALSE 2 GCCGTGCCGTAG 2269 pLX_TRC313_   924 GCTATCCGGCTATCCGG LacZ (1:100) pLas + 20170 T1 H01 pL42_ 97.10% GCTGCTTGCGATFALSE 2 ATCAGTGGCAGC 5905 pLX_TRC313_   924 pool1 ACCAATAG LacZ (1:100)pLas + 20170 T1 H01 sgRNA 88.90% GCTGCTTGCGAT FALSE 2 GCTGCTTGCGAT 1305pLX_TRC313_   924 ACCAATAG ACCAATAG LacZ (1:100) pLas + 20170 T1 H02pL42_ 47.60% GCTGCTTGCGAT TRUE 4 AACGATGGGACT 2653 pLX_TRC313_   924pool1 ACCAATAG LacZ (1:100) pLas + 20170 T1 H02 pL42_ 49.00% AGGAGGACGGCFALSE 4 TCATTCAGAGCG 2735 pLX_TRC313_   924 pool1 AACATCCTG LacZ (1:100)pLas + 20170 T1 H02 sgRNA 44.80% AAGGAGGACGG TRUE 4 AAGGAGGACGGC 1222pLX_TRC313_   924 CAACATCCT AACATCCT LacZ (1:100) pLas + 20170 T1 H02sgRNA 44.90% AGGAGGACGGC FALSE 4 AGGAGGACGGCA 1224 pLX_TRC313_   924AACATCCTG ACATCCTG LacZ (1:100) pLas + 20170 T1 H03 pL42_ 95.50%CAACATCCTGG FALSE 2 TCTTCACAACCG 5074 pLX_TRC313_   924 pool1 GGCACAAGCLacZ (1:100) pLas + 20170 T1 H03 sgRNA 90.00% CAACATCCTGG FALSE 2CAACATCCTGGG 4002 pLX_TRC313_   924 GGCACAAGC GCACAAGC LacZ (1:100)pLas + 20170 T1 H04 pL42_ 48.50% GTACAGCTAAG TRUE 3 CGTGTGATGATA 3104pLX_TRC313_   924 pool1 TTAAACTCG LacZ (1:100) pLas + 20170 T1 H04 pL42_48.60% AAGGAGGACGG FALSE 3 ATTGCTATTCGG 3108 pLX_TRC313_   924 pool1CAACATCCT LacZ (1:100) pLas + 20170 T1 H04 sgRNA 90.60% AAGGAGGACGGFALSE 3 AAGGAGGACGGC 3883 pLX_TRC313_   924 CAACATCCT AACATCCTLacZ (1:100)

SEQ ID NO: 1574 LOCUS pLas 7963 bp ds-DNA circular 01-FEB-2018DEFINITION . FEATURES Location/Qualifiersfeature    3591 . . . 4166           /label = “WPRE”feature           292 . . . 336               /label = “HIV-1_psi_pack”feature           1 . . . 181               /label = “HIV-1_5_LTR”promoter         1854 . . . 3111                /label = “EF-1a”misc_feature     1756 . . . 1841                /label =“scaffold_Dang_2015”misc_feature     3118 . . . 3492                /label = “ZeoR ”regulatory       846 . . . 1079               /label = “RRE”primer           1657 . . . 1676                /label = “LKO1_5_primer”feature          4302 . . . 4482                /label = “HIV-1_5_LTR”feature          4249 . . . 4301                /label = “delta_U3”variation        2242 . . . 2242                /label =“C in all sequences”feature          4232 . . . 4253                /label = “U3PPT”misc_feature     3516 . . . 3527                /label = “barcode”misc_feature     1736 . . . 1755                /label = “sgRNA”feature          4232 . . . 4247                /label = “cPPT”misc_feature     241 . . . 280               /label = “DIS_1”promoter         1495 . . . 1734                /label = “hU6_promoter”ORIGIN   1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca  61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc1501 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat1561 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt1621 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact1681 tgaaagtatt tcgatttctt ggctttatat atcttGTGGA AAGGACGAAA CACCgnnnnn1741 nnnnnnnnnn nnnnngtttC agagctaTGC TGGAAACAGC Atagcaagtt Gaaataaggc1801 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cTTTTTTgga tcctgcaaag1861 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa1921 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc1981 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt2041 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc2101 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac2161 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg2221 cgtgccttga attacttcca ctggctgcag tacgtgattc ttgatcccga gcttcgggtt2281 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga2341 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc2401 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg2461 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg2521 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg2581 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct2641 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc2701 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc2761 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa2821 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc2881 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg2941 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg3001 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc3061 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg atgtacaATG3121 GCCAAGTTGA CCAGTGCCGT TCCGGTGCTC ACCGCGCGCG ACGTCGCCGG AGCGGTCGAG3181 TTCTGGACCG ACCGGCTCGG GTTCTCCCGG GACTTCGTGG AGGACGACTT CGCCGGTGTG3241 GTCCGGGACG ACGTGACCCT GTTCATCAGC GCGGTCCAGG ACCAGGTGGT GCCGGACAAC3301 ACCCTGGCCT GGGTGTGGGT GCGCGGCCTG GACGAGCTGT ACGCCGAGTG GTCGGAGGTC3361 GTGTCCACGA ACTTCCGGGA CGCCTCCGGG CCGGCCATGA CCGAGATCGG CGAGCAGCCG3421 TGGGGGCGGG AGTTCGCCCT GCGCGACCCG GCCGGCAACT GCGTGCACTT CGTGGCCGAG3481 GAGCAGGACT GAgCTAGCtg ttcaatcaac attccNNNNN NNNNNNNact ggctattcat3541 tcgcCCTTTG GGTAAGCACA CGTCGAATTC GATATCAAGC TTATCGGTAA tcaacctctg3601 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta3661 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt3721 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc3781 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt3841 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg3901 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac3961 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc4021 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac4081 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct4141 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt4201 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa4261 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc4321 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa4381 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga4441 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat4501 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga4561 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa4621 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt4681 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc4741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga4801 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg4861 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta4921 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc4981 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg5041 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg5101 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct5161 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg5221 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag5281 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg5341 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc5401 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat5461 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag5521 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt5581 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa5641 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt5701 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt5761 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt5821 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg5881 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga5941 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa6001 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga6061 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa6121 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca6181 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta6241 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac6301 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc6361 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag6421 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga6481 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt6541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata6601 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag6661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa6721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt6781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc6841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa6901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa6961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc7021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa7081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa7141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg7201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc7261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg7321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg7381 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg7441 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat7501 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg7561 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt7621 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg7681 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta7741 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt7801 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc7861 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca7921 ttgcagagat attgtattta agtgcctagc tcgatacata aac //

REFERENCES

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Example 2

An example method of constructing libraries of genetic elements. ProvideA provides an example method of libraries comprising two engineeredassociations. The lentiviral vector shown under “mRNA contains barcode”(panel A) is an example of a vector with two elements (sgRNA andbarcode) that will normally undergo swapping unless the co-packagingprotocol is used. The associations did not undergo swapping because thelibraries were constructed using the co-packaging methods describedherein. The scatter plot (panel B) shows the accuracy of mapping over1000 barcodes to two categories. The mapped barcodes did not haverecombination, indicating the accuracy was improved by the co-packagingprotocol disclosed herein.

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A non-naturally occurring engineered lentiviralor retroviral system comprising a pooled set of payload polynucleotides,each having or encoding at least a genetic perturbation in associationwith a barcode that identifies the genetic perturbation(s), amultiplicity of carrier polynucleotides that are heterologous to thepayload polynucleotides and do not have or encode at least a geneticperturbation in association with a barcode that identifies the geneticperturbation(s), and one or more packaging polynucleotides, wherein thesystem comprises a 5:1 weight ratio or greater mixture of the carrierpolynucleotides to the pooled set of the payload polynucleotides,wherein a multiplicity of packaging cells transfected with the systemare capable of producing a viral expression library comprising viralparticles, wherein each one of the pooled set of the payloadpolynucleotides is comprised in at least one of the viral particles, andwherein the packaging cells are sufficiently capable of packaging nomore than one payload polynucleotide per viral particle such that targetcells transduced with the viral expression library have reducedrecombination activity, or template switching activity, or multipleintegration activity as compared to counterpart packaging cellstransfected with a mixture comprising the pooled set of the payloadpolynucleotides in the absence of the 5:1 weight ratio or greater of thecarrier polynucleotides to the pooled set of the payloadpolynucleotides.
 2. The engineered system of claim 1, wherein the systemcomprises a weight ratio of the carrier polynucleotides to the payloadpolynucleotides of 5:1 or greater, 10:1 or greater, 20:1 or greater,30:1 or greater, 40:1 or greater, 50:1 or greater, 60:1 or greater, 70:1or greater, 80:1 or greater, 90:1 or greater, 100:1 or greater, 200:1 orgreater, 300:1 or greater, 400:1 or greater, 500:1 or greater, 600:1 orgreater, 700:1 or greater, 800:1 or greater, 900:1 or greater, 1000:1 orgreater, 2000:1 or greater, 3000:1 or greater, 4000:1 or greater, or5000:1 or greater.
 3. The engineered system of claim 1, wherein eachcarrier polynucleotide comprises or encodes non-recombinogenic RNAsequences or proteins that are capable of dimerizing with the payloadpolynucleotides, and wherein the packaging cells are capable ofpackaging each payload polynucleotide as a dimer with one or more ofsaid carrier polynucleotides, non-recombinogenic RNA sequences, orproteins, such that target cells transduced with the viral expressionlibrary.
 4. The engineered system of claim 1, wherein each carrierpolynucleotide comprises single and/or double stranded DNA; recombinantand/or non-recombinant plasmid type vectors; replicable and/ornon-replicable plasmid type vectors; integrating and/or non-integratingplasmid type vectors; viral and/or non-viral plasmid type vectors;lentiviral or non-lentiviral plasmid type vectors; and/or retroviraland/or non-retroviral plasmid type vectors.
 5. The engineered system ofclaim 4, wherein the carrier polynucleotide comprises a plasmid selectedfrom the group consisting of pUC19, pr_H2b-BFB, pLX_TRC131_LacZ, andpR_LG.
 6. The engineered system of claim 1, wherein the geneticperturbation(s) is/are an over expressed gene, RNAi based system, a zincfinger nuclease, a transcription activator-like effector nuclease(TALEN), a meganuclease, or a CRISPR-Cas system, a component thereof, ora portion thereof.
 7. The engineered system of claim 6, wherein thepayload polynucleotides encode a CRISPR-Cas system or a componentthereof.
 8. The engineered system of claim 7, wherein the CRISPR-Cassystem is a CRISPR-Cas9 system.
 9. The engineered system of claim 8,wherein the payload polynucleotides encode one or more guide sequences.10. A method of screening cells for at least a genetic perturbationcomprising: providing a target cell or population of target cells in oneor more discrete volumes; introducing a viral expression librarycomprising viral particles, wherein the viral expression library isproduced by a multiplicity of packaging cells transfected with thesystem of claim 1, such that each target cell receives one of the set ofthe payload polynucleotides each having or encoding at least a geneticperturbation in association with a barcode that identifies the geneticperturbation(s); detecting genomic, genetic, proteomic, epigeneticand/or phenotypic differences in single cells; and identifying the atleast a genetic perturbation in each cell based on the associatedbarcode that identifies the genetic perturbation(s).
 11. A method ofpreparing one or more packaging cells capable of producing a viralexpression library the method comprising transfecting one or morepackaging cells with a system comprising a pooled set of payloadpolynucleotides, each payload polynucleotide comprising or encoding atleast a genetic perturbation in association with a barcode thatidentifies the genetic perturbation(s), a multiplicity of carrierpolynucleotides that are heterologous to the payload polynucleotides anddo not have or encode at least a genetic perturbation in associationwith a barcode that identifies the genetic perturbation(s), and one ormore packaging polynucleotides, wherein the system comprises a 5:1weight ratio or greater mixture of the carrier polynucleotides to thepooled set of the payload polynucleotides, wherein the packaging cellsare capable of producing a viral expression library comprising viralparticles, wherein each one of the pooled set of the payloadpolynucleotides is comprised in at least one of the viral particles ofthe viral expression library, wherein the packaging cells aresufficiently capable of packaging no more than one payloadpolynucleotide per viral particle such that target cells transduced withthe viral expression library have reduced recombination activity, ortemplate switching activity, or multiple integration activity, ascompared to counterpart packaging cells transfected with a mixturecomprising the payload polynucleotides in the absence of the 5:1 weightratio or greater of the carrier polynucleotides to the pooled set of thepayload polynucleotides.
 12. The method of claim 11, wherein the carrierpolynucleotide comprises or encodes non-recombinogenic RNA sequences orproteins that are capable of dimerizing with the payloadpolynucleotides, and wherein the packaging cells are capable ofpackaging each payload polynucleotide as a dimer with one or more ofsaid carrier polynucleotides, non-recombinogenic RNA sequences, orproteins, such that target cells transduced with the viral expressionlibrary.
 13. The method of claim 11, wherein the payload polynucleotidesencode an RNAi based system, a zinc finger nuclease, a transcriptionactivator-like effector nuclease (TALEN), a meganuclease, or aCRISPR-Cas system, or a component thereof.
 14. A viral expressionlibrary comprising viral particles obtained by transfecting thenon-naturally occurring engineered lentiviral or retroviral system ofclaim 1 into a multiplicity of packaging cells.
 15. A packaging cell orpackaging cells for producing a viral expression library comprisingviral particles, wherein one or more packaging cells comprise a systemcomprising a pooled set of payload polynucleotides, each payloadpolynucleotide comprising or encoding at least a genetic perturbationassociated with a barcode that identifies the genetic perturbation(s), amultiplicity of the carrier polynucleotides that are heterologous to thepayload polynucleotide and do not comprise or encode at least a geneticperturbation associated with a barcode that identifies the geneticperturbation(s), and one or more packaging polynucleotides, wherein thesystem comprises a 5:1 weight ratio or greater mixture of the carrierpolynucleotides to the pooled set of the payload polynucleotides,wherein the packaging cells are capable of producing a viral expressionlibrary comprising viral particles, wherein each one of the pooled setof the payload polynucleotides is comprised in at least one of the viralparticles of the viral expression library, and wherein the packagingcells are sufficiently capable of packaging no more than one payloadpolynucleotide per viral particle such that target cells transduced withthe viral expression library have reduced recombination activity, ortemplate switching activity, or multiple integration activity, ascompared to counterpart packaging cells transfected with a mixturecomprising the payload polynucleotides in the absence of the 5:1 weightratio or greater of the carrier polynucleotides to the pooled set of thepayload polynucleotides.
 16. A method of reducing intermolecularrecombination with a lentiviral genome plasmid of interest in a pooledlibrary, wherein the lentiviral genome plasmid of interest encodes atleast a genetic perturbation associated with a barcode that identifiesthe genetic perturbation(s), the method comprising transfecting one ormore packaging cells with a system comprising the pooled library oflentiviral genome plasmids, a multiplicity of viral carrier plasmids,wherein the lentiviral carrier plasmids are heterologous to thelentiviral genome plasmids and do not encode at least a geneticperturbation associated with a barcode that identifies the geneticperturbation(s), and one or more lentiviral packaging plasmids, whereinthe system comprises a 5:1 weight ratio or greater of the lentiviralcarrier plasmids to the lentiviral genome plasmids, wherein thepackaging cells are capable of producing a lentiviral expression librarycomprising lentiviral particles, wherein each lentiviral genome plasmidof interest is comprised in at least one of the lentiviral particles,and wherein the packaging cells are sufficiently capable of packaging nomore than lentiviral genome plasmid per lentiviral particle such thattarget cells transduced with the lentiviral expression library havereduced recombination activity, or template switching activity, ormultiple integration activity, as compared to counterpart packagingcells transfected with a mixture comprising the library of thelentiviral genome plasmids in the absence of the 5:1 weight ratio orgreater of the lentiviral carrier plasmids to the lentiviral genomeplasmids.
 17. The method of claim 16, wherein the lentiviral carrierplasmid comprises a non-integrating lentiviral vector.
 18. The method ofclaim 17, wherein the lentiviral carrier plasmid comprises anon-recombinogenic lentiviral vector.
 19. The method of claim 16,wherein the weight ratio of the lentiviral carrier plasmid to thelentiviral genome plasmid is at least 10:1.
 20. The method of claim 16,wherein the weight ratio of the lentiviral carrier plasmid to thelentiviral genome plasmid is at least 50:1.
 21. The method of claim 16,wherein the weight ratio of the lentiviral carrier plasmid to thelentiviral genome plasmid is at least 100:1.
 22. The method of claim 16,wherein the library comprises a barcode library.
 23. The method of claim16, wherein the library comprises a plurality of guide polynucleotides.24. The method of claim 16, wherein the library comprises a plurality ofsgRNAs.